Modified biotin-binding protein, fusion proteins thereof and applications

ABSTRACT

The disclosure provides modified biotin-binding proteins which can be expressed in soluble form in high yield in bacteria. Also provided are fusion proteins comprising the modified biotin-binding protein and an antigen. The disclosure further provides non-hemolytic variants of alpha-hemolysin from S. aureus and fusion protein comprising non-hemolytic variant of alpha-hemolysin and a biotin-binding domains. Immunogenic compositions comprising the proteins are also disclosed and use of such immunogenic compositions for inducing an immune response or for vaccinating a subject are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/992,343 filed May 30, 2018, which is a continuation application ofU.S. Ser. No. 14/946,372 filed Nov. 19, 2015, which is a divisionalapplication of U.S. Ser. No. 14/116,492 filed Jan. 27, 2014, now U.S.Pat. No. 9,499,593 issued Nov. 22, 2016, which application is a 35U.S.C. § 371 National Phase Entry Application of InternationalApplication No. PCT/US2012/037541 filed May 11, 2012, which designatesthe U.S., and which claims benefit under 35 U.S.C. § 119(e) of the U.S.Provisional Application Ser. No. 61/484,934 filed May 11, 2011, U.S.Provisional Application Ser. No. 61/608,168, filed Mar. 8, 2012, andU.S. Provisional Application Ser. No. 61/609,974, filed Mar. 13, 2012,the contents of each of which is are incorporated fully herein byreference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant NumbersA1066013 and AI067737 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 21, 2022, isnamed 701039-074341 _SL.TXT and is 58,009 bytes in size.

TECHNICAL FIELD

The present disclosure relates to biotin-binding proteins and the fusionproteins/compositions comprising such biotin-binding proteins. Alsodescribed herein are methods for expressing biotin-binding proteinsand/or the fusion proteins thereof in high yield and in soluble inbacteria.

BACKGROUND

Biotin-binding protein and their derivatives can be widely used invarious applications. However, production or purification of recombinantbiotin-binding proteins can be very difficult. When expressed in E.coli, most biotin-binding proteins tend to accumulate in inclusionbodies, denaturing, refolding and tedious downstream processing arerequired in the preparation of active proteins. Expression in E. coli ispreferable because of the low cost of production and the potential forfurther engineering; thus, biotin-binding proteins that can beefficiently produced in E. coli are highly sought after. Further,expression E. coli also provides the possibility to generate recombinantfusion proteins containing biotin-binding protein for variousapplications.

Accordingly, there is need in the art for biotin-binding proteins andfusion proteins containing biotin-binding proteins which can beexpressed in soluble form in high yields in E. coli.

SUMMARY

One objective of the present disclosure is to provide a recombinantbiotin-binding protein, which can be expressed in soluble form in highyields in E. coli. Accordingly, the present disclosure providesbiotin-binding proteins and compositions comprising the same. In someembodiments, the recombinant biotin-binding protein comprises an E. colisignal sequence fused to the N-terminus of an amino acid sequencecomprising amino acids 45-179

SEQ ID NO: 1) (FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKD,of wild-type Rhizavidin (rhavi). In some embodiments, the E. coli signalsequence is MKKIWLALAGLVLAFSASA (SEQ ID No: 2). The signal sequence canbe fused with the sequence comprising amino acids 45-179 of wild-typerhavi by a flexible peptide linker.

Provided herein is also a method of expressing a biotin-binding proteinin soluble form in high yield in E. coli. In some embodiments, themethod comprising expressing a biotin-binding protein in E. coli,wherein the native signal sequence of the biotin-binding protein hasbeen replaced by an E. coli signal sequence. In some embodiments, thesignal sequence is MKKIWLALAGLVLAFSASA (SEQ ID No: 2)

In yet another aspect, the invention provides biotin-binding fusionprotein comprising a biotin-binding domain and a protein or a peptide.

In another aspect provided herein is a lipidated biotin-binding protein.As used herein, the term “lipidated biotin-binding protein” refers to abiotin-binding protein that is covalently linked with a lipid. Thelipidated biotin-binding proteins are ligands or agonists of Toll likereceptor 2. Accordingly, also provided herein are methods for inducingan immune response in subject. The method comprising administering tothe subject a composition comprising a lipidated biotin-binding protein.

Provided herein is also a method of expressing a lipidatedbiotin-binding protein in E. coli. In some embodiments, the methodcomprises expressing a lipidated biotin-binding protein in E. coli,wherein the native signal sequence of the biotin-binding protein hasbeen replaced by an E. coli signal sequence containing a lipidationmotif. In some embodiments, the signal sequence is

(SEQ ID No: 3) MKKVAAFVALSLLMAGC

In still another aspect, provided herein is a non-hemolytic derivativeof Staphylococcal aureus alpha-hemolysin (Hla). The Hla derivativedescribed herein can be in the form of a fusion protein, wherein thefusion protein comprises both the Hla derivative domain and abiotin-binding domain. In some embodiments of this aspect, thebiotin-binding domain is a biotin-binding protein described herein.

Like the lipidated biotin-binding proteins, the Hla variants or theirfusion proteins with biotin-binding proteins described herein are alsoligands or agonists of Toll like receptors or other pattern recognitionreceptors (PRRs). Accordingly, also provided herein are methods forinducing an immune response in subject. In some embodiments, the methodcomprising administering to the subject a composition comprising anon-hemolytic Hla variants or their fusion proteins with biotin-bindingprotein described herein.

In yet still another aspect, provided herein is an immunogeniccomposition or vaccine composition comprising a biotin-binding protein,a lipidated biotin-binding protein, a biotin-binding fusion proteincomprising a biotin-binding domain and an antigenic protein or peptide.In some embodiments of this aspect, the antigenic protein is anon-hemolytic derivative of Hla described herein.

Provided herein also is a method of vaccinating a subject, e.g., amammal, e.g., a human with the immunogenic compositions as disclosedherein, the method comprising administering a vaccine composition asdisclosed herein to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a modified biotin-bindingprotein according to an embodiment disclosed herein.

FIG. 2 is a SDS-PAGE of a purified recombinant biotin-binding proteindescribed herein.

FIG. 3 is a schematic representation of a fusion protein comprising abiotin-binding protein and an antigen X

FIG. 4 is an exemplary SDS-PAGE of purified rhizavidin fusion proteins

FIG. 5 is a schematic representation of lipidated biotin-binding proteinaccording to an embodiment disclosed herein.

FIG. 6 is a SDS-PAGE of a lipidated biotin-binding protein describedherein.

FIG. 7 is a bar graph showing dose-dependent TLR2 activity of thelipidated biotin-binding protein.

FIG. 8 is a schematic representation of recombinant WT or mutant S.aureus alpha-hemolysin (Hla) and their rhizavidin fusion proteinsdescribed herein. In the non-hemolytic Hla, point mutations were made asfollow: (i) residue 205 W to A; (ii) residue 213 W to A; or (iii)residues 209-211 from DRD to AAA.

FIG. 9 is a SDS-PAGE of purified wild-type Hla or non-hemolytic variantsthereof.

FIG. 10 is SDS-PAGE of purified biotin-binding fusion proteins ofwild-type Hla or non-hemolytic variants thereof.

FIG. 11 is a line graph showing hemolytic activity of WT Hla andnon-hemolytic variants thereof.

FIG. 12 is a line graph showing hemolytic activity of wild-type Hla,non-hemoytic variants of Hla, and biotin-binding fusion proteins ofwild-type Hla and non-hemoytic variants of thereof.

FIG. 13 is a bar graph showing that stimulation of macrophages withbiotin-binding fusion protein (rhavi-Hla209) induces multiplepro-inflammatory cytokines.

FIG. 14 is a bar graph showing that multiple antigen presenting system(MAPS) complex containing lipidated rhizavidin or rhavi-Hla209 inducesstronger T cell responses to the antigens.

FIG. 15 shows a hierarchal schematic table and showing a variety ofdifferent embodiments of the MAPS platform, showing that the MAPScomposition can be designed and manufactured to elicit a particular,broad spectrum, or variety of antigenic targets.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

It should be understood that this invention is not limited to theparticular composition, methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims.

Without wishing to be bound by a theory, low expression of abiotin-binding protein in the art can be due to bad-folding caused bythe disulfide bond in each monomer of the biotin-binding protein whichdoes not form, or forms at very low levels, in the cytoplasm of E. coli.Now the inventors have discovered that correct-folding can be achievedby transporting into the periplasmic space of E. coli. Thus, correctfolding of recombinant biotin-binding proteins can be improved byreplacing the complete native signal sequence of a biotin-bindingprotein with an E. coli secretion signal sequence. Without wishing to bebound by a theory, this facilitates the translocation of recombinantprotein into the periplasmic space of E. coli cells. Translocation ofrecombinant protein into the periplasmic space of E. coli then canprovide the functionally important disulfide bond in the biotin-bindingprotein (e.g., in Rhizavidin) and the protein can fold correctly in asoluble form and in high yields.

In one aspect, provided herein is a biotin-binding protein that can beexpressed in a soluble form and high yield in E. coli. As used herein,the term “biotin-binding protein” refers to a protein, whichnon-covalently binds to biotin or an analogue or derivative thereof.High yield means that the protein can be expressed in a soluble form inE. coli at an amount of about 10 mg/L, 11 mg/L, 12 mg/L, 13 mg/L, 14mg/L, 15 mg/L, 20 mg/L, 25 mg/L, 30 mg/L, 35 mg/L, 30 mg/L, 35 mg/L, 40mg/L, 45 mg/L, 50 mg/L or more.

In some embodiments, the biotin-binding protein can be a recombinantprotein. The coding sequence for the biotin-binding protein can beoptimized using E. coli expression codons, to avoid any difficultyduring expression in E. coli due to rare codons present in originalgene.

Generally, the biotin-binding protein comprises a biotin-binding domain.As used herein, a “biotin-binding domain” refers to a polypeptidesequence that binds to biotin. While a complete biotin-binding proteincan be used as a biotin-binding domain, only the biotin-binding portionof the protein can be used. In some embodiments, the biotin-bindingdomain is from Rhizavidin.

In some embodiments, the biotin-binding domain consists of, or consistsessentially of, the amino acid sequence corresponding to amino acids45-179 of the wild-type Rhizavidin. Amino acid sequence of the wild-typeRhizavidin is:

(SEQ ID NO: 4) MIITSLYATFGTIADGRRTSGGKTMIRTNAVAALVFAVATSALAFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKD

In other words, the biotin-binding domain does not comprise (i.e.,lacks) lacks amino acids 1-44(MIITSLYATFGTIADGRRTSGGKTMIRTNAVAALVFAVATSALA, SEQ ID NO: 5). of thewild-type Rhizavidin. In some embodiments, the biotin-binding domaincomprises the amino acid sequence

(SEQ ID NO: 1) FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKD.

In some embodiments, the biotin-binding domain comprises an amino acidsequence having at least 50% identity, at least 55% identity, at least60% identity, at least 65% identity, at least 70% identity, at least 75%identity, at least 80% identity, preferably at least 85% identity, atleast 90% identity, at least 95% identity, at least 96% identity, atleast 97% identity, at least 98% identity, or at least 99% identity, andmore preferably at least 99.3% identity to SEQ ID NO: 1).

While, Helppolainen et al. (Biochem J., 2007, 405: 397-405) describeremoving only first 24 residues of the full length Rhizavidin, theinventors have discovered that the first 44 residues of full lengthRhizavidin are unnecessary for the core structure and function ofRhizavidin. Further, unexpectedly, amino acids 25-44(MIRTNAVAALVFAVATSALA, SEQ ID NO: 6) of the full length Rhizavidinreduce the solubility and secretion of Rhizavidin expressed in E. colias replacement of the first 44 residues of full length Rhizavidin withan E. coli signal peptide led to an increase in the solubility andsecretion in E. coli of biotin proteins described herein.

In the biotin-binding protein described herein, the biotin-bindingdomain can be extended on the N- or C-terminus by one or more aminoacids with the proviso that the N-terminus of the biotin-binding domaindoes not comprise an amino acid sequence corresponding to an amino acidsequence 1-44 of the wild-type Rhizavidin. The inventors have discoveredthat truncating the first 44 amino acids on the N-terminus of the wildtype Rhizavidin can dramatically increase expression of thebiotin-binding protein in soluble form in E. coli. Thus, thebiotin-binding protein described herein can comprise the sequenceX¹-X²-X³, wherein X² is a peptide having the amino acid sequencecorresponding to amino acids 45-179 of the wild-type Rhizavidin and X¹and X³ are independently absent or a peptide of 1 to about 100 aminoacids with the proviso that the N-terminus of X¹ does not comprise anamino acid sequence corresponding to N-terminus of amino acids 1-44 ofthe wild-type Rhizavidin.

In some embodiments, the biotin-binding proteins can comprise a signalpeptide conjugated to the N-terminus of the biotin-binding protein, i.e.X¹ can comprise a signal peptide. The signal peptide is also called aleader peptide in the N-terminus, which may or may not be cleaved offafter the translocation through the membrane. Secretion/signal peptidesare described in more detail below. In some embodiments, the signalsequence is MKKIWLALAGLVLAFSASA (SEQ ID NO: 2), MAPFEPLASGILLLLWLIAPSRA(SEQ ID NO: 7), MKKVAAFVALSLLMAGC (SEQ ID NO: 3), or a derivative orfunctional portion thereof.

The signal peptide can be linked to the N-terminus of the biotin-bindingdomain either directly (e.g., via a bond) or indirectly (e.g., by alinker). In some embodiments, the signal peptide can be linked to theN-terminus of the biotin-binding domain by a peptide linker. The peptidelinker sequence can be of any length. For example, the peptide linkersequence can be one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen or more amino acids inlength. In some embodiments, the peptide linker is four amino acids inlength.

The peptide linker sequence can comprise any amino acid sequence. Forexample, the peptide linker can comprise an amino acid sequence whichcan be cleaved by a signal peptidase. In some embodiments, the peptidelinker comprises the amino acid sequence AQDP (SEQ ID NO: 8) or VSDP(SEQ ID NO: 9).

In the biotin-binding protein, the biotin-binding domain can beconjugated at its C-terminus to a peptide of 1-100 amino acids. Suchpeptides at the C-terminus can be used for purification tags, linkers toother domains, and the like.

In some embodiments, the biotin-binding protein comprises on its N- orC-terminus one or more (e.g., one, two, three, four, five, six, seven,eight, nine, ten or more) purification tags. Examples of purificationtags include, but are not limited to a histidine tag, a c-my tag, a Halotag, a Flag tag, and the like. In some embodiments, the biotin-bindingprotein comprises on its C-terminus a histidine tag, e.g. a (His)₆ (SEQID NO. 10).

A purification tag can be conjugated to the biotin-binding protein by apeptide linker to enhance the probability that the tag is exposed to theoutside. The length of the linker can be at least one (e.g., one, two,three, four, five six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, or fifteen) amino acid. The linker peptide cancomprise any amino acid sequence without limitations. In someembodiments, the linker peptide comprises the amino acid sequenceVDKLAAALE (SEQ ID NO: 11) or GGGGSSSVDKLAAALE (SEQ ID NO: 12).

In some embodiments, the biotin-binding protein comprises on itsC-terminus the amino acid sequence VDKLAAALEHHHHH (SEQ ID NO: 13) orGGGGSSSVDKLAAALEHHHHHH (SEQ ID NO: 14).

In some embodiments, the biotin-binding protein comprises the amino acidsequence:

(SEQ ID NO: 15) MKKIWLALAGLVLAFSASAAQDPFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSVDKLAAALEHHHHHH.

Compared with known biotin-binding proteins which form tetramers, thebiotin-binding protein described herein form a dimer. Without wishing tobe bound by a theory, forming a dimer can further improve expression ofthe biotin-binding protein described herein as a soluble protein in E.coli.

Although biotin-binding proteins are known in the art, thebiotin-binding protein described herein comprises significantdifferences from avidin and avidin-like proteins currently known in theart. First, currently known avidins are quite difficult to express assoluble proteins in E. coli. However, as the inventors havedemonstrated, the biotin-binding protein described herein can beexpressed as a soluble protein in E. coli in high yield.

The biotin-binding proteins described herein can be obtained in asoluble form in high yields, e.g., over 30 mg per liter of culture, byexpression in E. coli. Thus, the biotin-binding proteins describedherein are more soluble than those described in the art and reflectunderlying differences. Without wishing to be bound by a theory, thedifference in solubility can be attributed to underlying physical and/orchemical and/or structural differences between biotin-binding proteinsdescribed herein and other biotin-binding proteins known in the art.

Second, the biotin-binding protein described herein comprises abiotin-binding domain which consists of amino acids 45-179 of wild-typeRhizavidin. While, wild-type Rhizavidin and a partially truncatedportion thereof are known in the art, there is no teaching or suggestionin the art that a biotin-binding protein comprising the amino acidssequence of amino acids 45-179 of wild-type Rhizavidin and having an E.coli signal sequence would lead to a soluble protein that can beobtained in high yield in E. coli. According to Helppolainen et al.(Biochem J., 2007, 405: 397-405) the amino acids 25-44 of the wild typeRhizavidin comprise a putative signal sequence. However, as discussesherein, the inventors have discovered and demonstrated that replacementof the putative signal sequence with an E. coli signal sequence leads toincrease in soluble form of the biotin-binding protein expression in E.coli.

Third, the biotin-binding protein described comprises a peptide of aminoacid sequence GGGGSSSVDKLAAALEHHHHHH (SEQ ID NO: 14). This peptide atthe C-terminus provides a histidine tag for purification and a place forinsertion of other domains, e.g. antigenic domains, in the biotinprotein. Further, while Helppolainen et al. (Biochem J., 2007, 405:397-405) describe expression of Rhizavidin in E. coli, there is noteaching or suggestion in Helppolainen et al. for conjugating anadditional peptide to the C-terminus of the biotin-binding domain ofRhizavidin.

Fourth, Rhizavidin has a lower sequence homology to egg avidin (22.4%sequence identity and 35.0% similarity) compared with other avidin-likeproteins. Thus, the biotin-binding protein described herein is differentavidin and other avidin-like proteins.

Fifth, the biotin-binding protein described herein has a low isoelectricpoint (pI) compared to the avidin and other avidin-like molecules. Theisoelectric point of the wild type Rhizavidin is 4.0 (Helppolainen etal., Biochem J., 2007, 405: 397-405). The isoelectric point of otherknown biotin-binding proteins is generally over 6.1 (see Helppolainen etal., Biochem J., 2007, 405: 397-405). In comparison, the pI of thebiotin-binding protein described herein is 5.4. The acidic pI of thebinding-protein described herein leads to reduced non-specific binding.A problem in the use of currently known avidin and avidin-like peptidesis non-specific binding thereof. Currently known avidin and avidin-likepeptides can non-specifically bind to not only cells but also DNAs,proteins, and biological materials such as membranes. For example, indetection of a material using the avidin-biotin binding, avidinnon-specifically binds to materials other than the object material to bedetected to increase the background. One reason for the highnon-specific binding of avidin include its high isoelectric point.Avidin is a strongly basic protein, having a significantly highisoelectric point of 10 or more, and is positively charged as a whole.Accordingly, it is believed that avidin readily binds to biologicalmaterials, which are negatively charged in many cases. Thus, the low pIof the biotin-binding protein described herein is advantageous over thecurrently known avidin and avidin-like peptides.

Sixth, size of the biotin-binding protein described herein is arelatively small compared to currently known avidin and avidin-likeproteins. The biotin-binding protein is smaller than 28 kDa (dimersize). However, most of the currently known avidin and avidin-likeproteins all have sizes larger than 60 kDa (tetramer size). Wild-typeRhizavidin is said to be about 29 kDa (dimer size) in size. Small sizeof the biotin-binding protein can be used to increase loading of bindingconjugation between molecules interest. For example, the biotin-bindingprotein can be used to conjugate first molecule of interest with asecond molecule of interest. One of the molecules of interest can belinked to one or more biotin or biotin-like molecules and the secondmolecule can be linked, conjugated or fused to the biotin-bindingprotein. Given the small size of the biotin-protein described herein,the biotin or biotin-like molecules can be spaced closer together on theto permit binding of more relative to if the second molecule was alarger currently known avidin or avidin-like molecule.

Seventh, the biotin-binding protein described herein is a dimer. Forminga dimer can further improve expression of the biotin-binding proteindescribed herein as a soluble protein in E. coli. Additionally, becausethe biotin-binding protein forms a dimer rather than tetramer like allother known avidin-like proteins, 1) the structural complexity of thefusion antigens is reduced; 2) the difficulty of expressing recombinantbiotin-binding protein fusion proteins is similarly reduced, 3) thesteric hindrance of manipulations of biotin-binding protein fusions isminimized, which is advantageous for further manipulations with, forexample, but not limited to, biotin, biotin mimetics or biotinderivatives, and 4) solubility of biotin-binding protein fusions isgreatly enhanced. Thus, demonstrating underlying differences between thebiotin-binding proteins described herein and those known in the art.

Eighth, the biotin-binding protein described herein reduces the risk ofan immunogenic composition comprising the same inducing an egg-relatedallergic reaction in a subject. Moreover, antibody to biotin-bindingdomain described herein has no apparent cross-reactivity to egg avidin(and vice versa).

Further, a biotin-binding protein described herein can have improvedproperties, such as a reduction in non-specific binding or a furtherimprovement in biotin binding, while retaining the characteristics ofwild-type Rhizavidin. The use of the biotin-binding protein describedherein for detection, for example, in immunoassay or nucleic acidhybridization assay, for measuring an analyte utilizing avidin-biotinbinding can reduce background, increase sensitivity, and maintain thebinding property with biotin in severe conditions.

This study clearly demonstrates the advantages and differences of thebiotin-binding proteins described herein over avidin and otheravidin-like proteins. Thus, the biotin-binding proteins described hereinhave a potential as a powerful and versatile tool in a wide range ofapplications utilizing avidin-biotin technology.

Without limitations, a biotin-binding protein can be used in anymethodology, composition, or system requiring the use of anavidin-biotin system. As one of ordinary skill is well aware, theavidin-biotin system can be used for numerous laboratory methods, suchas bioconjugation; target molecule detection; target molecule isolation,purification, or enrichment from a sample; protein detection; nucleicacid detection; protein isolation, purification, or enrichment; nucleicacid isolation, purification, or enrichment; ELISA; flow cytometry; andthe like.

Accordingly, exemplary uses for the recombinant biotin-binding proteinsdescribed herein include, but are not limited to, bioconjugation; targetmolecule detection; target molecule isolation, purification, orenrichment from a sample; protein detection; nucleic acid detection;protein isolation, purification, or enrichment; nucleic acid isolation,purification, or enrichment; ELISA; flow cytometry; and the like.

In some embodiments, the biotin-binding protein described herein can beused as part of the affinity pair in the multiple antigen presentingsystem (MAPS) as described in U.S. Provisional application No.61/48,934, filed May 11, 2012; No. 61/608,168, filed Mar. 8, 2012; andNo. 61/609,974, filed Mar. 13, 2012, and PCT application no.PCT/US12/37412, filed May 11, 2012, content of all of which isincorporated herein by reference in its entirety. MAPS is also describedin more detail herein below. Without wishing to be bound by a theory,use of a biotin-binding protein described herein reduces the risk of theMAPS inducing an egg-related allergic reaction in a subject. Moreover,antibody to recombinant modified Rhizavidin has no apparentcross-reactivity to egg avidin (and vice versa).

Lipidated Biotin-Binding Protein

In another aspect provided herein is a lipidated biotin-binding protein.As used herein, the term “lipidated biotin-binding protein” refers to abiotin-binding protein that is covalently conjugated with a lipid. Thelipid moieties could be a diacyl or triacyl lipid.

The lipidated biotin-binding protein can be made using a lipidationsequence. As used herein, the term “lipidation sequence” refers to anamino acid sequence that facilitates lipidation in a bacteria, e.g., E.coli, of a polypeptide carrying the lipidating sequence. The lipidationsequence can be present at the N-terminus or the C-terminus of theprotein. The lipidation sequence can be linked to the recombinantbiotin-binding protein to form a fusion protein, which is in lipidatedform when expressed in E. coli by conventional recombinant technology.In some embodiments, a lipidation sequence is located at the N-terminusof the biotin-binding protein.

Any lipidation sequence known to one of ordinary skill in the art can beused. In some embodiments, the lipidating sequence is MKKVAAFVALSLLMAGC(SEQ ID NO: 3) or a derivative or functional portion thereof. Otherexemplary lididationg sequences include, but are not limited to,MNSKKLCCICVLFSLLAGCAS (SEQ ID NO: 16), MRYSKLTMLIPCALLLSAC (SEQ ID NO:17), MFVTSKKMTAAVLAITLAMSLSAC (SEQ ID NO: 18), MIKRVLVVSMVGLSLVGC (SEQID NO: 19), and derivatives or functional portions thereof.

In some embodiments, the lipidation sequence can be fused to thebiotin-binding protein via a peptide linker, wherein the peptide linkerattaches the lipidating sequence to the biotin-binding protein.

In some embodiment, the peptide linker comprises the amino acid sequenceVSDP (SEQ ID NO: 9).

In one embodiment, the biotin-binding lipid protein comprises the aminoacid sequence

(SEQ ID NO: 20) MKKVAAFVALSLLMAGCVSDPFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTEN KSLLKD.

The lipidated biotin-binding proteins are ligands for Toll LikeReceptors (TLRs). As such, the lipidated biotin-binding proteinsdescribed herein can be used as TLR ligands. For example, the lipidatedbiotin-binding protein can be used in compositions to induce TLR2stimulation. This can be useful for inducing immunogenicity to otherantigens/pathogens. Thus, the biotin-binding lipoproteins can be used inimmunogenic compositions as a co-stimulation factor or adjuvant for anantigen.

As used herein, the term “Toll Like Receptor” is meant to refer ingeneral to any Toll-like receptor of any species of organism. A TLR canbe from any mammalian species. TLRs have been identified in variousmammalian species including, but not limited to, for example, humans,guinea pigs, and mice. A specific TLR can be identified with additionalreference to species of origin (e.g., human, murine, etc. . . . ), aparticular receptor (e.g., TLR2, TLR3, TLR9, etc. . . . ), or both. Insome embodiments, the lipidated biotin-binding protein is a ligand forTLR2.

Toll-like receptors (TLRs) are a family of germline encodedtransmembrane proteins that facilitate pathogen recognition andactivation of the innate immune system. Toll-like receptors (TLRs) arepattern recognition receptors (PRRs), and are expressed by cells of theinnate immune system, including macrophages, dendritic cells and NKcells. Examples of known ligands for TLRs include gram positive bacteria(TLR-2), bacterial endotoxin (TLR-4), flagellin protein (TLR-5),bacterial DNA (TLR-9), double-stranded RNA and poly I:C (TLR-3), andyeast (TLR-2). Other ligands that bind an endocytic pattern recognitionreceptor, a scavenger receptor or a mannose-binding receptor can also becontemplated by the instant invention. TLRs engage conservedpathogen-derived ligands and subsequently activate the TLR/IL-1R signaltransduction pathway to induce a variety of effector genes. Toll-likereceptors (TLRs) represent an important group of PRRs that can sensepathogen- or microbe-associated molecular patterns. They are widelyexpressed in blood, spleen, lung, muscle and intestines by many types ofcells, notably dendritic cells (DCs) but also macrophages, epithelialcells, and lymphocytes.

Whereas some TLRs located on the cell surface are specific for microbiallipids and proteins, others associated with endosomal compartmentsinside cells are specific for nucleic acids. Ligation of the TLRs bytheir specific ligands results in conformational changes in thereceptors, leading to downstream signal transduction that primarilyinvolves MyD88- and TRIF-dependent pathways. Except for TLR3, all otherTLRs can signal through the MyD88 pathway to induce pro-inflammatorycytokines that involve activation of intracellular protein kinasecascades including IB kinase (IKK)—NF—B, and extracellular signalregulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38mitogen-activation protein kinases (MAPKs). The TRIF pathway,independent of MyD88, is utilized by both TLR3 and TLR4 and mediates theinduction of type I interferons.

The recombinant biotin-binding lipoproteins described herein haveenhanced immunogenicity. Without wishing to be bound by a theory, lipidmoieties at the N-terminals of the lipoproteins or lipopeptidescontribute to the adjuvant activity. Accordingly, additional embodimentsprovide immunogenic or vaccine compositions for inducing animmunological response, comprising the isolated biotin-bindinglipoprotein, or a suitable vector for in vivo expression thereof, orboth, and a suitable carrier, as well as to methods for eliciting animmunological or protective response comprising administering to a hostthe isolated recombinant biotin-binding lipoprotein, the vectorexpressing the recombinant biotin-binding lipoprotein, or a compositioncontaining the recombinant lipoprotein or vector, in an amountsufficient to elicit the response.

An immunological or immunogenic composition comprising thebiotin-binding lipoprotein elicits an immunological response—local orsystemic. The response can, but need not, be protective. It is to benoted that as used herein, the terms “immunological composition” and“immunogenic composition” include a “vaccine composition” (as the twoformer terms can be protective compositions). Without limitations, alipidated biotin-binding protein described herein can be used as anantigen, adjuvant, or a co-stimulator in an immunological, immunogenic,or vaccine composition. Further, since the lipidated biotin-bindingprotein comprises a biotin-binding domain, the lipidated protein can beassembled to the polymer backbone of the MAPS. Accordingly, providedherein are also methods of inducing an immunological response in a hostmammal. The method comprising administering to the host an immunogenic,immunological or vaccine composition comprising a lipidatedbiotin-binding protein described herein and a pharmaceuticallyacceptable carrier or diluent.

In some embodiments, the lipidated biotin-binding protein is a fusionprotein comprising a lipidated biotin-binding protein and a protein orpeptide.

Non-Hemolytic Hla

Hemolysins are exotoxins produced by bacteria that cause lysis of redblood cells. While highly immunogenic, their use in vaccines is limitedbecause they cause lysis of red blood cells. Accordingly, in anotheraspect, provided herein are variants of staphylococcal aureusalpha-hemolysin (Hla), its fusion construct with biotin-binding proteinand its uses. These variants, designated herein as “mHla,” havesubstantially non-hemolytic, i.e., have substantially low hemolyticactivity. As used herein, the phrase “substantially non-hemolytic” meansan inability to lyse red blood cells at equivalent titers of wild-typeHla. The term “wild-type Hla” is accorded the usual definitionassociated with such phrase, i.e., Hla that is naturally secreted by acapable bacterial source. “Wild-type Hla,” by definition, does notinclude, e.g., Hla fusion products derived via recombinant DNAtechniques. In some embodiments, hemolytic activity of mHla is at least5%, at least 10%, at least 15%, at least 20%, at least 20%, at least30%, at least 30%, at least 35%, least 40%, at least 45%, at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% lower than anequivalent titers of wild-type Hla (SEQ ID NO: 59), wherein the aminoacid sequence of SEQ ID NO: 59 corresponds to amino acids 27-319 of thewild-type Hla mature polypeptide. In some embodiments, the mHla has nodetectable hemolytic activity. The inventors have also discovered thathemolytic activity of mHla can be further reduced by linking the mHlawith a biotin-binding protein. Accordingly, the present disclosure alsodescribes fusion proteins comprising a mHla protein and a biotin-bindingprotein.

As provided herein, a non-hemolytic Hla can be created wherein residueW205 or W213 is substituted with alanine (A) or the tripeptideDRD209-211 is substituted with a tri-alanine peptide (AAA) in thewild-type Hla. The mutated Hla protein can be expressed and purified inan E. coli expression system. The mutants can be made by point mutationusing quick change mutagenesis. For example, the nucleotide sequence ofa nucleic acid encoding the wild-type Hla can be changed to replace agiven amino acid in the wild-type Hla to another amino acid.

In some embodiments, the mHla is a fusion protein comprising the mHlaand a biotin-binding protein. In some embodiments, the biotin-bindingmHla fusion protein comprises the amino acid sequence

(SEQ ID NO: 21) MKKIWLALAGLVLAFSASAAQDPFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYAAASWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKID WEKEEMTN.

The Hla variants described herein are ligands for Toll Like Receptors(TLRs). As such, the Hla variants described herein can be used as TLRligands. For example, the Hla variants can be used in compositions toinduce TLR2 stimulation. This can be useful for inducing immunogenicityto other antigens/pathogens. Thus, the Hla variants described herein canbe used in immunogenic compositions as a co-stimulation factor oradjuvant for an antigen. Further, when the mHla is fused with abiotin-binding protein, the fusion protein can be conjugated to thepolymer backone of the MAPS.

In some embodiments, the mHla can be used as a co-stimulatory factor inan immunogenic or vaccine composition.

Further, since the mHla induce an immune response in the subject, themHla can be used as in an immunogenic or vaccine composition forvaccinating a subject against S. aureus.

The mHla described herein have enhanced immunogenicity. Accordingly,additional embodiments provide immunogenic or vaccine compositions forinducing an immunological response, comprising the mHla, or a suitablevector for in vivo expression thereof, or both, and a suitable carrier,as well as to methods for eliciting an immunological or protectiveresponse comprising administering to a host the isolated mHla, thevector expressing the mHla, or a composition containing the mHla orvector, in an amount sufficient to elicit the response.

An immunological or immunogenic composition comprising the mHla canelicit an immunological response—local or systemic. The response can,but need not, be protective. Accordingly, a non-hemolytic mutant of Hladescribed herein can be as an antigen, adjuvant, or a co-stimulator inan immunological, immunogenic, or vaccine composition.

Further, provided herein are also methods of inducing an immunologicalresponse in a host mammal. The method comprising administering to thehost an immunogenic, immunological or vaccine composition comprising anon-hemolytic mutant of Hla described herein and a pharmaceuticallyacceptable carrier or diluent.

In another aspect, provided herein are fusion proteins comprising abiotin-binding protein described herein linked to an antigenic proteinor peptide. These fusion proteins are also referred to as biotin-bindingfusion proteins and as antigen fusion proteins herein. Thebiotin-binding protein and the antigenic protein or peptide can belinked in any configuration, e.g., biotin-binding protein can in theN-terminal and the antigenic peptide in the C-terminal of the fusionprotein or vice versa.

In some embodiments, the biotin-binding protein and the antigenicprotein or peptide are linked to each other by a peptide linker. Withoutlimitations, the peptide linker can comprise any amino acid sequence andcan be of any length. For example, the peptide linker sequence can beone, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen or more amino acids in length. Insome embodiments, the peptide linker linking the antigen domain to thebiotin-binding domain is eight amino-acids in length.

In some embodiments, the peptide linker linking the antigen domain tothe biotin-binding domain has the amino acids sequence GGGGSSS (SEQ IDNO: 22).

In some embodiments, the antigenic protein is a non-hemolytic Hladescribed herein.

In some embodiments, the non-hemolytic Hla protein is a fusion proteincomprising a biotin-binding protein and a non-hemolytic Hla describedherein.

In some embodiments, the non-hemolytic Hla protein is a fusion proteincomprising a lipidated biotin-binding protein and a non-hemolytic Hladescribed herein.

Immunogenic Compositions

In another aspect, provided herein are immunogenic compositionscomprising an antigen fusion protein, a lipidated biotin-binding, or anon-hemolytic variant of Hla described herein. In addition, providedherein are also immunogenic compositions and vaccine compositionscomprising an immunogenic complex that comprises at least one antigenfusion protein, or multiple antigen fusion proteins, attached to apolymer scaffold for use in eliciting an immune response to each of theantigens attached to the polymer, and optionally to the polymer itself,when administered to a subject. Without wishing to be bound by a theory,the immunogenic composition described herein stimulates a humoral andcellular immune response: it can generate antibody and the Th1/Th17responses to multiple protein antigens using a single MAPS immunogenicconstruct. A combination of B- and T-cell immunity to the organismrepresents an optimal vaccine strategy against many diseases, includingpneumococcal disease associated invasive infection and nasopharyngealcarriage. In some embodiments, the immunogenic composition is a vaccineor is included in a vaccine.

Accordingly, the embodiments herein provide for an immunogeniccomposition and methods useful for raising an immune response in asubject, which can be used on its own or in conjunction or admixturewith essentially any existing vaccine approaches.

In some embodiments, an immunogenic composition as disclosed hereincomprises at least 2 antigens, or at 3 least antigens, or at least 5antigens, or between 2-10 antigens, or between 10-15 antigens, orbetween 15-20 antigens, or between 20-50 antigens, or between 50-100antigens, or more than 100 antigens, inclusive. In some embodiments,where an immunogenic composition as disclosed herein comprises at least2 antigens, the antigens can be the same antigen or at least 2 differentantigens. In some embodiments, the antigens can be from the same ordifferent pathogens, or can be different epitopes or parts of the sameantigenic protein, or can be the same antigen which is specific todifferent serotypes or seasonal variations of the same pathogen (e.g.,influenza virus A, B, and C).

In some embodiments, an immunogenic composition as disclosed hereincomprises an antigen from a pathogenic organism or an abnormal tissue.In some embodiments, the antigen is a tumor antigen. In someembodiments, an antigen can be at least one antigen selected fromantigens of pathogens or parasites, such as antigens of Streptococcuspneumoniae, Mycobacterium tuberculosis or M. tetanus, Bacillusanthracis, HIV, seasonal or epidemic influenza antigens (such as H1N1 orH5N1), Bordetella pertussis, Staphylococcus aureus, Neisseriameningitides or N. gonorrhoeae, HPV, Chlamydia trachomatis, HSV or otherherpes viruses, or Plasmodia sp. These antigens may include peptides,proteins, glycoproteins, or polysaccharides. In some embodiments, theantigen is a toxoid or portion of a toxin.

In some embodiments, an immunogenic composition as disclosed hereincomprises an antigenic polysaccharide, for example, such as Vi antigen(Salmonella typhi capsular polysaccharide), pneumococcal capsularpolysaccharides, pneumococcal cell wall polysaccharide, Hib (Haemophilusinfluenzae type B) capsular polysaccharide, meningococcal capsularpolysaccharides, and other bacterial capsular or cell wallpolysaccharides, or any combinations thereof. The polysaccharide mayhave a protein component, e.g., a glycoprotein such as those fromviruses.

In some embodiments, an immunogenic composition as disclosed hereinfurther comprises at least one co-stimulation factor associated with thepolymer or polysaccharide, where the co-stimulation factor can beassociated directly or indirectly. For example, in some embodiment, aco-stimulation factor can be covalently attached to the polymer. Forexample, in some embodiments, a co-stimulation factor can be covalentlyattached to the first affinity molecule, which is then cross-linked tothe polymer. For example, in some embodiments, a co-stimulation factorcan be attached to a complementary affinity molecule, which associateswith a first affinity molecule to link the co-stimulator factor to thepolymer. In some embodiments, a co-stimulation factor is an adjuvant. Inalternative embodiments, a co-stimulatory factor can be any known to oneof ordinary skill in the art, and includes any combination, for example,without limitation, Toll like receptor agonists (agonists for TLR2, 3,4, 5 7, 8, 9, etc.), NOD agonists, or agonists of the inflammasome.

In some embodiments, the co-stimulatory factor can be a lipidatedbiotin-binding protein or a non-hemolytic variant of alpha-hemolysin orthe fusion protein of mHla with biotin-binding protein described herein.

Another aspect of the present invention relates to the use of theimmunogenic composition as disclosed herein to be administered to asubject to elicit an immune response in the subject. In someembodiments, the immune response is an antibody/B cell response, a CD4⁺T-cell response (including Th1, Th2 and Th17 cells) and/or a CD8⁺ T-cellresponse. In some embodiments, at least one adjuvant is administered inconjunction with the immunogenic composition.

Another aspect of the present invention relates to a method for inducingan immune response in a subject to at least one antigen, comprisingadministering to the subject the immunogenic composition as disclosedherein.

Another aspect of the present invention relates to a method ofvaccinating an animal, e.g., a bird, a mammal or a human, against atleast one antigen comprising administering a vaccine compositioncomprising the immunogenic composition as disclosed herein.

In all aspects as disclosed herein, an animal or a subject can be ahuman. In some embodiments, the subject can be an agricultural ornon-domestic animal, or a domestic animal. In some embodiments, avaccine composition comprising the immunogenic composition as disclosedherein can be administered via subcutaneous, intranasal, oral,sublingual, vaginal, rectal, intradermal, intraperitoneal, or intramuscular injection.

In all aspects as disclosed herein, an immune response is anantibody/B-cell response, a CD4⁺ T-cell response (including Th1, Th2 andTh17 responses) or a CD8+ T-cell response against protein/peptideantigen(s). In some embodiments, an immune response is anantibody/B-cell response against the polymer, e.g., a pneumococcalpolysaccharide. In some embodiments, at least one adjuvant isadministered in conjunction with the immunogenic composition.

Another aspect of the present invention relates to the use of theimmunogenic composition as disclosed herein for use in a diagnostic forexposure to a pathogen or immunogenic agent.

Multiple Antigen Presenting System

Also provided herein is also an immunogenic multiple antigen presentingsystem (MAPS), useful for the production of immunogenic compositions,such as those useful in vaccines. In particular, the present inventionrelates to compositions comprising an immunogenic complex comprising atleast one type of polymer, e.g., a polysaccharide, that can, optionally,be antigenic; at least one antigenic protein or peptide; and at leastone complementary affinity-molecule pair comprising (i) a first affinitymolecule that associates with the polymer, and (ii) a complementaryaffinity molecule that associates with the protein or peptide; such thatthe first and complementary affinity molecules serve as an indirect linkbetween the polymer with the antigenic protein or peptide. Accordingly,the polymer can attach at least 1, or at least 2, or a plurality of thesame or different protein or peptide antigens. In some embodiments, thepolymer is antigenic, e.g., the polymer is a pneumococcal capsularpolysaccharide. In some embodiments, the protein or peptide antigens arerecombinant protein or peptide antigens.

The immunogenic compositions as disclosed herein can elicit both humoraland cellular responses to one or multiple antigens at the same time. Theimmunogenic compositions provide for a long-lasting memory response,potentially protecting a subject from future infection. This allows fora single immunogenic composition that raises a high titer of functionalanti-polysaccharide antibody, and is similar or compares favorably withthe antibody level induced by conventional conjugate vaccine. Moreover,there is no restriction to specific carrier protein, and various antigenproteins can be used in MAPS construct to generate a robustanti-polysaccharide antibody response.

Additionally, the strong antibody response and Th17/Th1 responses arespecific to multiple protein antigens presented via the MAPScomposition. This presents a major advantage, as a means for elicitingtwo forms of immunity with one construct. In addition to a moreconventional immune response to an antigenic polysaccharide conjugatedto a protein carrier, the present invention provides for a T-cellresponse and, more specifically, Th17 and Th1 responses to proteinsinjected systemically. Moreover, the present immunogenic composition canincorporate ligands onto the polymer backbone. This provides a potentialto enhance specific B-cell or T-cell responses by modifyingprotein/polymer ratio, complex size, or by incorporating specificco-stimulatory factor, such as TLR2/4 ligands, etc., into thecomposition.

Compared with typical conjugation technology, which involves harshtreatment of proteins, the present methods avoid risk of denaturation ofother modification of the peptide antigen. This provides a substantialadvantage of preserving the antigenicity of the included proteins andincreases the probability that the protein itself will serve as anantigen (rather than just a carrier). Similarly, the present methodsavoid unnecessary modification/damage of the polysaccharide backbone,because there is no heavy chemical cross-linking: biotinylation can beprecisely controlled to react with specific functional groups of thepolysaccharide, and the biotinylation level can be easily adjusted. Thisis advantageous in avoiding the typical process of conjugation, thatresults in damage to critical side chains or epitopes, which may causereduced immunogenicity and protection.

The present the affinity-based assembly provides easy and highlyflexible preparation of the immunogenic composition. It is highlyspecific and stable; it can remain in the cold for months and retain itspotency. The assembly process is simple enough to ensure highreproducibility; there are only a few steps required, which reduces therisk of lot-to-lot variation, of great industrial advantage. The MAPSassembly is highly efficient (over 95%), even at low concentrations ofprotein and polysaccharide (such as 0.1 mg/ml); this is a majoradvantage, because inefficiencies in conjugate manufacture (typicallyefficiencies are in the <50% range) represent a major hurdle and reasonfor the high cost of vaccines. For formulation: it is easy to adjust thecomposition and physical properties of the final product. Theprotein:polymer ratio in the complex is adjustable; with moderatebiotinylation of polymer, protein:polymer can be 10:1 (w/w) or more;conversely, the ratio can be 1:10 or less if such is the interest basedon immunological goals. Additionally, the size of the immunogenic MAPScomposition can be adjusted by the choice of polymer size. The methodsof making the MAPS provide for ease in combining proteins and polymerswith little modification. The possible multivalency of final product byloading multiple protein antigens, from the same or different pathogens(e.g., pneumococcus and tuberculosis), in single immunogenic construct,provides for a composition that can be used to decrease the number ofvaccines required to immunize a subject against more than one disease.Moreover, the MAPS composition is highly stable; becoming dissociatedonly upon boiling and maintaining immunogenicity even after many monthsat 4° C. The immunogenicity of the MAPS complex may be limited by thestability of the antigenic protein or peptide component, which stabilitymay be extended by inclusion in the MAPS complex. The specific antigensused herein exhibited stability at room temperature and after at leastone freeze-thaw cycle. This provides an important advantage over currentvaccines that are compromised if the “cold chain” is not maintainedcarefully.

Accordingly, one aspect of the present invention relates to animmunogenic composition comprising a polymer, at least one protein orpeptide antigen, and at least one complementary affinity-molecule pair,where the complementary affinity-molecule pair comprises a firstaffinity molecule that associates with the polymer and a complementaryaffinity molecule that associates with the protein or peptide antigen,so that when the first affinity molecule associates with thecomplementary affinity molecule, it indirectly links the antigen to thepolymer.

In some embodiments, the first affinity molecule is cross-linked to thepolymer with a cross-linking reagent, for example, a cross-linkingreagent selected from CDAP(1-cyano-4-dimethylaminopyridiniumtetrafluoroborate), EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimidehydrochloride), sodium cyanoborohydride; cyanogen bromide; or ammoniumbicarbonate/iodoacetic acid. In some embodiments, the first affinitymolecule is cross-linked to carboxyl, hydroxyl, amino, phenoxyl,hemiacetal, and mecapto functional groups of the polymer. In someembodiments, the first affinity molecule is covalently bonded to thepolymer.

In some embodiments, the first affinity molecule is biotin or aderivative thereof, or a molecule with similar structure or physicalproperty as biotin, for example, an amine-PEG3-biotin((+)-biotinylation-3-6,9-trixaundecanediamine) or derivative thereof.

In some embodiments, the protein or peptide antigen of the immunogeniccomposition is a fusion protein comprising the antigenic protein orpeptide fused to the complementary affinity binding molecule. The fusioncan be a genetic construct, i.e., a recombinant fusion peptide orprotein. In some embodiments, an antigen can be covalently attached as afusion protein to the complementary affinity molecule. In alternativeembodiments, the antigen is non-covalently attached to the complementaryaffinity molecule.

In some embodiments, the complementary affinity molecule is abiotin-binding protein or a derivative or a functional portion thereof.In some embodiments, a complementary affinity molecule is an avidin-likeprotein or a derivative or a functional portion thereof, for example butnot limited to, rhizavidin or a derivative thereof. In some embodiments,a complementary affinity molecule is avidin or streptavidin or aderivative or a functional portion thereof.

In some embodiments, a secretion signal peptide is located at theN-terminus of the avidin-like protein. Any signal sequence known topersons of ordinary skill in the art can be used; and in someembodiments, the signal sequence is MKKIWLALAGLVLAFSASA (SEQ ID NO: 2)or a derivative or functional portion thereof. In some embodiments, theantigen can be fused to a complementary affinity molecule via a flexiblelinker peptide, where the flexible linker peptide attaches the antigento the complementary affinity molecule.

In some embodiments, the polymer component of the immunogen comprises apolymer derived from a living organism, e.g., a polysaccharide. In someembodiments, a polymer can be purified and isolated from a naturalsource, or is can be synthesized as with a naturalcomposition/structure, or it can be a synthetic (e.g., with anartificial composition/structure) polymer. In some embodiments, apolymer is derived from an organism selected from the group consistingof: bacteria, archaea, or eukaryotic cells like fungi, insect, plant, orchimeras thereof. In some embodiments, the polymer is a polysaccharidederived from a pathogenic bacterium. In specific embodiments thepolysaccharide is a pneumococcal capsular polysaccharide, a pneumococcalcell-wall polysaccharide, or a Salmonella typhi Vi polysaccharide.

In some embodiments, a polymer of the immunogenic composition asdisclosed herein is branched chain polymer, e.g., a branchedpolysaccharide, or alternatively, can be a straight chain polymer, e.g.,a single chain polymer, e.g., polysaccharide. In some embodiments, thepolymer is a polysaccharide, for example, dextran or a derivativethereof. In some embodiments, a polymer, e.g., dextran polysaccharidecan be of average molecular weight of 425 kD-500 kDa, inclusive, or insome embodiments, greater than 500 kDa. In some embodiments, a polymer,e.g., dextran polysaccharide can be of average molecular weight of60kD-90 kDa, inclusive, or in some embodiments, smaller than 70 kDa. Thedextran polymer can be derived from a bacterium, such as Leuconostocmesenteroides.

In some embodiments, an immunogenic composition as disclosed hereincomprises at least 2 antigens, or at 3 least antigens, or at least 5antigens, or between 2-10 antigens, or between 10-15 antigens, orbetween 15-20 antigens, or between 20-50 antigens, or between 50-100antigens, or more than 100 antigens, inclusive. In some embodiments,where an immunogenic composition as disclosed herein comprises at least2 antigens, the antigens can be the same antigen or at least 2 differentantigens. In some embodiments, the antigens can be from the same ordifferent pathogens, or can be different epitopes or parts of the sameantigenic protein, or can be the same antigen which is specific todifferent serotypes or seasonal variations of the same pathogen (e.g.,influenza virus A, B, and C).

In some embodiments, an immunogenic composition as disclosed hereincomprises an antigen from a pathogenic organism or an abnormal tissue.In some embodiments, the antigen is a tumor antigen. In someembodiments, an antigen can be at least one antigen selected fromantigens of pathogens or parasites, such as antigens of Streptococcuspneumoniae, Mycobacterium tuberculosis or M. tetanus, Bacillusanthracis, HIV, seasonal or epidemic influenza antigens (such as H1N1 orH5N1), Bordetella pertussis, Staphylococcus aureus, Neisseriameningitides or N. gonorrhoeae, HPV, Chlamydia trachomatis, HSV or otherherpes viruses, or Plasmodia sp. These antigens may include peptides,proteins, glycoproteins, or polysaccharides. In some embodiments, theantigen is a toxoid or portion of a toxin.

In some embodiments, an immunogenic composition as disclosed hereincomprises an antigenic polysaccharide, for example, such as Vi antigen(Salmonella typhi capsular polysaccharide), pneumococcal capsularpolysaccharides, pneumococcal cell wall polysaccharide, Hib (Haemophilusinfluenzae type B) capsular polysaccharide, meningococcal capsularpolysaccharides, the polysaccharide of Bacillus anthracis (the causativeagent of anthrax), and other bacterial capsular or cell wallpolysaccharides, or any combinations thereof. The polysaccharide mayhave a protein component, e.g., a glycoprotein such as those fromviruses.

In some embodiments, an immunogenic composition as disclosed hereinfurther comprises at least one co-stimulation factor associated with thepolymer or polysaccharide, where the co-stimulation factor can beassociated directly or indirectly. For example, in some embodiment, aco-stimulation factor can be covalently attached to the polymer. Forexample, in some embodiments, a co-stimulation factor can be covalentlyattached to the first affinity molecule, which is then cross-linked tothe polymer. For example, in some embodiments, a co-stimulation factorcan be attached to a complementary affinity molecule, which associateswith a first affinity molecule to link the co-stimulation factor to thepolymer. In some embodiments, a co-stimulation factor is an adjuvant. Inalternative embodiments, a co-stimulatory factor can be any known to oneof ordinary skill in the art, and includes any combination, for example,without limitation, Toll like receptor agonists (agonists for TLR2, 3,4, 5 7, 8, 9, etc.), NOD agonists, or agonists of the inflammasome.

Another aspect of the present invention relates to the use of theimmunogenic composition as disclosed herein to be administered to asubject to elicit an immune response in the subject. In someembodiments, the immune response is an antibody/B cell response, a CD4⁺T-cell response (including Th1, Th2 and Th17 cells) and/or a CD8⁺ T-cellresponse. In some embodiments, at least one adjuvant is administered inconjunction with the immunogenic composition.

Another aspect of the present invention relates to a method for inducingan immune response in a subject to at least one antigen, comprisingadministering to the subject the immunogenic composition as disclosedherein.

Another aspect of the present invention relates to a method ofvaccinating an animal, e.g., a bird, a mammal or a human, against atleast one antigen comprising administering a vaccine compositioncomprising the immunogenic composition as disclosed herein.

In all aspects as disclosed herein, an animal or a subject can be ahuman. In some embodiments, the subject can be an agricultural ornon-domestic animal, or a domestic animal. In some embodiments, avaccine composition comprising the immunogenic composition as disclosedherein can be administered via subcutaneous, intranasal, oral,sublingual, vaginal, rectal, intradermal, intraperitoneal, intramuscular injection, or via skin-patch for transcutaneous immunization.

In all aspects as disclosed herein, an immune response is anantibody/B-cell response, a CD4⁺ T-cell response (including Th1, Th2 andTh17 responses) or a CD8+ T-cell response against protein/peptideantigen(s). In some embodiments, an immune response is anantibody/B-cell response against the polymer, e.g., a pneumococcalpolysaccharide. In some embodiments, at least one adjuvant isadministered in conjunction with the immunogenic composition.

Another aspect of the present invention relates to the use of theimmunogenic composition as disclosed herein for use in a diagnostic forexposure to a pathogen or immunogenic agent.

Provided herein also is a method of vaccinating a subject, e.g., amammal, e.g., a human with the immunogenic compositions as disclosedherein, the method comprising administering a vaccine composition asdisclosed herein to the subject.

Generally, the immunogenic compositions and compositions comprising animmunogenic complex can comprise at least one antigen, or multipleantigens, attached to a polymer scaffold for use in eliciting an immuneresponse to each of the antigens attached to the polymer, and optionallyto the polymer itself, when administered to a subject. This multipleantigen presenting system (MAPS), stimulates a humoral and cellularimmune response: it can generate anti-polysaccharide antibody and theB-cell/Th1/Th17 responses to multiple protein antigens using single MAPSimmunogenic construct. A combination of B- and T-cell immunity to theorganism might represent an optimal vaccine strategy against manydiseases, including pneumococcal disease associated invasive infectionand nasopharyngeal carriage. In some embodiments, the immunogeniccomposition is a vaccine or is included in a vaccine.

Accordingly, one aspect of the present invention relates to animmunogenic composition (multiple antigen presenting system, or MAPS)comprising at least one polymer, e.g., one polysaccharide, at least oneprotein or peptide antigen, and at least one complementaryaffinity-molecule pair comprising (i) a first affinity moleculeassociated with the polymer, and (ii) a complementary affinity moleculeassociated with the antigen, which serves to indirectly attach theantigen to the polymer (e.g., the first affinity molecule associateswith the complementary affinity molecule to link the antigen to thepolymer). Accordingly, as the polymer can be used as a scaffold toattach at least 1, or at least 2, or a more (e.g., a plurality) of thesame or different antigens. The immunogenic compositions as disclosedherein can be used to elicit both humoral and cellular immunity tomultiple antigens at the same time.

Accordingly, the embodiments herein provide for an immunogeniccomposition and methods useful for raising an immune response in asubject, which can be used on its own or in conjunction or admixturewith essentially any existing vaccine approaches.

The MAPS is a flexible and versatile composition that can be designedand manufactured to elicit a particular, broad spectrum, or variety ofantigenic targets. FIG. 15 is a hierarchal schematic and provides asimple example guide for envisioning the flexibility of MAPSembodiments.

Polymers

One component of MAP consists of a “backbone,” typically a polymer. Thepolymer may be antigenic or non-antigenic. It can be made of a widevariety on substances, as described herein, with the caveat that thepolymer serves as a means of presenting the associated antigen(s) to theimmune system in immunogenic fashion. In some embodiments, the polymeris a synthetic polymer. In some embodiments, the polymer is a naturallyoccurring polymer, e.g., a polysaccharide derived or purified frombacterial cells. In some embodiments, the polysaccharide is derived orpurified from eukaryotic cells, e.g., fungi, insect or plant cells. Inyet other embodiments, the polymer is derived from mammalian cells, suchas virus-infected cells or cancer cells. In general, such polymers arewell known in the art and are encompassed for use in the methods andcompositions as disclosed herein.

In some embodiments, a polymer is a polysaccharide selected from any ofthe following, dextran, Vi polysaccharide of Salmonella typhi,pneumococcal capsular polysaccharide, pneumococcal cell wallpolysaccharide (CWPS), meningococcal polysaccharide, Haemophilusinfluenzae type b polysaccharide, or any another polysaccharide ofviral, prokaryotic, or eukaryotic origin.

In some embodiments, the polysaccharide consists of or comprises anantigenic sugar moiety. For example, in some embodiments, apolysaccharide for use in the methods and immunogenic compositions asdisclosed herein is a Vi polysaccharide of Salmonella typhi. The Vicapsular polysaccharide has been developed against bacterial entericinfections, such as typhoid fever. Robbins et al., 150 J. Infect. Dis.436 (1984); Levine et al., 7 Baillieres Clin. Gastroenterol. 501 (1993).Vi is a polymer of α-1→4-galacturonic acid with an N acetyl at positionC-2 and variable 0-acetylation at C-3. The virulence of S. typhicorrelates with the expression of this molecule. Sharma et al., 101 PNAS17492 (2004). The Vi polysaccharide vaccine of S. typhi has severaladvantages: Side effects are infrequent and mild, a single dose yieldsconsistent immunogenicity and efficacy. Vi polysaccharide may bereliably standardized by physicochemical methods verified for otherpolysaccharide vaccines, Vi is stable at room temperature and it may beadministered simultaneously with other vaccines without affectingimmunogenicity and tolerability. Azze et al., 21 Vaccine 2758 (2003).

Thus, the Vi polysaccharide of S. typhi may be cross-linked to a firstaffinity molecule as disclosed herein, for attaching at least oneantigen to the polysaccharide. In some embodiments, the antigen can befrom the same or from another organism, such that the resultingimmunogenic composition confers at least some level of immunity againstone pathogen, or two different pathogens: if the antigen confersprotection against pneumococcus, an immunogenic composition where thepolymer scaffold is a Vi polysaccharide can raise an immunogenicresponse against both S. typhi and pneumococci. Other examples includecombining sugars from encapsulated bacteria (such as meningococcus, S.aureus, pneumococcus, Hib, etc.) and tuberculosis antigens, to providean immunogenic composition that raises an immune reponse against twodifferent pathogens.

Other polysaccharide (PS) moieties that may be used in the presentinvention in alternative to dextran, bacterial cell wall polysaccharides(CWPS), etc., include carbohydrate antigens of cancers.

Further in regard to pneumococcal polysaccharides, the polysaccharidecan be derived from any of the over 93 serotypes of pneumococcus thathave been identified to date, for example, including but not limited toserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F,14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Additional serotypesmay be identified and included in the present immunogenic composition asdescribed herein. More than one pneumococcal polysaccharide can beincluded as the polymer backbone of the present immunogenic compositionsor in a vaccine comprising the present MAPS compositions.

The polysaccharide can also be derived from the invention, theimmunogenic composition comprises N. meningitidis capsularpolysaccharides from at least one, two, three or four of the serogroupsA, C, W, W135, or Y.

A further embodiment comprises the Type 5, Type 8, or any of thepolysaccharides or oligosaccharides of Staphylococcus aureus.

In some embodiments, the polymer is chimeric polymer comprising morethan one type of polymer. For example a polymer of the immunogeniccomposition as disclosed herein can comprise a portion of polymer A, andthe remaining portion of polymer B. There is no limit to the amount ofdifferent types of polymers which can be used in a single MAPS backboneentity. In some embodiments, where the polymer is a branched polymer,the chain polymer can be polymer A, and the branches can be at least 1or at least 2 or at least 3 or more different polymers.

In some embodiments, the polymer is a branched polymer. In someembodiments, the polymer is a single chain polymer.

In some embodiments, the polymer is a polysaccharide comprising at least10 carbohydrate repeating units, or at least 20, or at least 50, or atleast 75, or at least 100, or at least 150, or at least 200, or at least250, or at least 300, or at least 350, or at least 400, or at least 450,or at least 500, or more than 500 repeating units, inclusive.

In one aspect of the invention, the polysaccharide (PS) can have amolecular mass of <500 kDa or >500 kDa. In another aspect of theinvention, the PS has a molecular mass of <70 kDa.

In some embodiments, a polymer is a large molecular weight polymer,e.g., a polymer can be of an average molecular weight of between about425-500 kDa, inclusive, for example, at least 300 kDa, or at least 350kDa, or at least 400 kDa, or at least 425 kDa, or at least 450 kDa, orat least 500 kDa or greater than 500 kDa, inclusive, but typically lessthan 500 kDa.

In some embodiments, a polymer can be a small molecular weight polymer,e.g., a polymer can be of an average molecular weight of between about60 kDA to about 90 kDa, for example, at least 50 kDa, or at least 60kDa, or at least 70 kDa, or at least 80 kDa, or at least 90 kDa, or atleast 100 kDa, or greater than 100 kDa, inclusive, but generally lessthan about 120 kDa.

In some embodiments, the polymer is harvested and purified from anatural source; and in other embodiments, the polymer is synthetic.Methods to produce synthetic polymers, including syntheticpolysaccharides, are known to persons of ordinary skill and areencompassed in the compositions and methods as disclosed herein.

Just a few of the polysaccharide polymers that can serve as a backbonefor one or more antigens or antigen types are exemplified in Table 1:

TABLE 1 Example polysaccharide polymer MAPS backbone and associatedexample antigens Protein Antigens Polysaccharide Number of antigensAntigen origins Dextran D90 (60-90 KD) two pneumococcus D150 (150 KD)three pneumococcus D270 (270 KD) three pneumococcus D500 (425-575 KD)two; three; six pneumococcus Pneumococcal Serotype 1 one, two, three,five pneumococcus, tuberculosis, capsular staphylococcus polysaccharideSerotype 3 five pneumococcus, tuberculosis Serotype 5 one; two; three;five pneumococcus, tuberculosis Serotype 6B two pneumococcus Serotype 7three pneumococcus Serotype 14 one; two; three; five pneumococcus,tuberculosis Serotype 19 three pneumococcus Pneumococcal cell wallpolysaccharide five pneumococcus S. typhi Vi polysaccharide fivepneumococcus

Additional polymers that can be used in the immunogenic MAPScompositions described herein include polyethylene glycol-basedpolymers, poly(ortho ester) polymers, polyacryl carriers, PLGA,polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimers, β-amino esterpolymers, polyphosphoester (PPE), liposomes, polymerosomes, nucleicacids, phosphorothioated oligonucleotides, chitosan, silk, polymericmicelles, protein polymers, virus particles, virus-like-particles (VLPs)or other micro-particles. See, e.g., El-Sayed et al., Smart PolymerCarriers for Enhanced Intracellular Delivery of Therapeutic Molecules, 5Exp. Op. Biol. Therapy, 23 (2005). Biocompatible polymers developed fornucleic acid delivery may be adapted for use as a backbone herein. See,e.g., BIOCOMPATIBLE POL. NUCL. ACID. DELIV. (Domb et al., eds., JohnWiley & Sons, Inc. Hoboken, N.J., 2011).

For example, VLPs resemble viruses, but are non-infectious because theydo not contain any viral genetic material. The expression, includingrecombinant expression, of viral structural proteins, such as envelopeor capsid components, can result in the self-assembly of VLPs. VLPs havebeen produced from components of a wide variety of virus familiesincluding Parvoviridae (e.g., adeno-associated virus), Retroviridae(e.g., HIV), and Flaviviridae (e.g., Hepatitis B or C viruses). VLPs canbe produced in a variety of cell culture systems including mammaliancell lines, insect cell lines, yeast, and plant cells. Recombinant VLPsare particularly advantageous because the viral component can be fusedto recombinant antigens as described herein.

Antigens

The fusion proteins and immunogenic compositions as disclosed herein cancomprise any antigen that elicits an immune response in a subject. Insome embodiments, at least one or more antigens are associated with thepolymer of the composition. In some embodiments, at least 2, or at least3, or at least 5, or at least 10, or at least 15, or at least 20, or atleast 50, or at least 100, or more than 100 antigens can be associatedwith the polymer as disclosed herein. In some embodiments, where theimmunogenic composition comprises more than one antigen, the antigenscan be the same antigen or they can be a variety of different antigensassociated with the polymer. In some embodiments, where the immunogeniccomposition comprises more than one antigen, the antigens can beantigens from the same pathogen or from different pathogens, oralternatively, can be different antigens from the same pathogen, orsimilar antigens from different serotypes of pathogens.

An antigen for use in the fusion proteins and immunogenic compositionsand methods described herein can be any antigen, including, but notlimited to pathogenic peptides, toxins, toxoids, subunits thereof, orcombinations thereof (e.g., cholera toxin, tetanus toxoid).

In some embodiments, an antigen, which can be fused to the complementaryaffinity molecule, can be any antigen associated with an infectiousdisease, or cancer or immune disease. In some embodiments, an antigencan be an antigen expressed by any of a variety of infectious agents,including virus, bacterium, fungus or parasite.

In some embodiments, an antigen is derived (e.g., obtained) from apathogenic organism. In some embodiments, the antigen is a cancer ortumor antigen, e.g., an antigen derived from a tumor or cancer cell.

In some embodiments, an antigen derived from a pathogenic organism is anantigen associated with an infectious disease; it can be derived fromany of a variety of infectious agents, including virus, bacterium,fungus or parasite.

In some embodiments, a target antigen is any antigen associated with apathology, for example an infectious disease or pathogen, or cancer oran immune disease such as an autoimmune disease. In some embodiments, anantigen can be expressed by any of a variety of infectious agents,including virus, bacterium, fungus or parasite. A target antigen for usein the methods and compositions as disclosed herein can also include,for example, pathogenic peptides, toxins, toxoids, subunits thereof, orcombinations thereof (e.g., cholera toxin, tetanus toxoid).

Non-limiting examples of infectious viruses include: Retroviridae;Picornaviridae (for example, polio viruses, hepatitis A virus;enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (such as strains that cause gastroenteritis); Togaviridae(for example, equine encephalitis viruses, rubella viruses); Flaviridae(for example, dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (for example, coronaviruses); Rhabdoviridae (forexample, vesicular stomatitis viruses, rabies viruses); Filoviridae (forexample, ebola viruses); Paramyxoviridae (for example, parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (for example, influenza viruses); Bungaviridae (forexample, Hantaan viruses, bunga viruses, phleboviruses and Nairoviruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g.,reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae(Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae(papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses);Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zostervirus, cytomegalovirus (CMV), Marek's disease virus, herpes viruses);Poxviridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (such as African swine fever virus); and unclassifiedviruses (for example, the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses). The compositions and methods described herein arecontemplated for use in treating infections with these viral agents.

Examples of fungal infections that may be addressed by inclusion ofantigens in the preaent embodiments include aspergillosis; thrush(caused by Candida albicans); cryptococcosis (caused by Cryptococcus);and histoplasmosis. Thus, examples of infectious fungi include, but arenot limited to, Cryptococcus neoformans, Histoplasma capsulatum,Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis,Candida albicans. Components of these organisms can be included asantigens in the MAPS described herein.

In one aspect of the invention, an antigen is derived from an infectiousmicrobe such as Bordatella pertussis, Brucella, Enterococci sp.,Neisseria meningitidis, Neisseria gonorrheae, Moraxella, typeable ornontypeable Haemophilus, Pseudomonas, Salmonella, Shigella,Enterobacter, Citrobacter, Klebsiella, E. coli, Helicobacter pylori,Clostridia, Bacteroides, Chlamydiaceae, Vibrio cholera, Mycoplasma,Treponemes, Borelia burgdorferi, Legionella pneumophilia, Mycobacteriasps (such as M. tuberculosis, M. avium, M. intracellulare, M. kansaii,M. gordonae, M. leprae), Staphylococcus aureus, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus anthracis, Corynebacteriumdiphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Leptospira sps., Pasteurella multocida,Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis,Treponema pallidium, Treponema pertenue, and Actinomyces israelli. Thecompositions and methods described herein are contemplated for use intreating or preventing infections against these bacterial agents.

Additional parasite pathogens from which antigens can be derivedinclude, for example: Entamoeba histolytica, Plasmodium falciparum,Leishmania sp., Toxoplasma gondii, Rickettsia, and the Helminths.

In another aspect of the invention, an antigen is a truncatedpneumococcal PsaA protein, pneumolysin toxoid pneumococcalserine/threonine protein kinase (StkP), pneumococcal serine/threonineprotein kinase repeating unit (StkPR), pneumococcal PcsB protein,staphylococcal alpha hemolysin, Mycobacterium tuberculosis mtb proteinESAT-6, M. tuberculosis cell wall core antigen, Chlamydia CT144, CT242or CT812 polypeptides or fragments of these, Chlamydia DNA gyrasesubunit B, Chlamydia sulfite synthesis/biphosphate phosphatase,Chlamydia cell division protein FtsY, Chlamydia methionyl-tRNAsynthetase, Chlamydia DNA helicase (uvrD), Chlamydia ATP synthasesubunit I (atpl), or Chlamydia metal dependent hydrolase.

An embodiment of the present invention provides for an immunogeniccomposition targeting the pathogen Myocobacterium tuberculosis (TB), anintracellular bacterial parasite. One example of a TB antigen is TbH9(also known as Mtb 39A). Other TB antigens include, but are not limitedto, DPV (also known as Mtb8.4), 381, Mtb41, Mtb40, Mtb32A, Mtb64, Mtb83,Mtb9.9A, Mtb9.8, Mtb16, Mtb72f, Mtb59f, Mtb88f, Mtb71f, Mtb46f andMtb31f, wherein “f” indicates that it is a fusion or two or moreproteins.

As noted above, an antigen can be derived from a Chlamydia species foruse in the immunogenic compositions of the present invention.Chlamydiaceae (consisting of Chlamydiae and Chlamydophila), are obligateintracellular gram-negative bacteria. Chlamydia trachomatis infectionsare among the most prevalent bacterial sexually transmitted infections,and perhaps 89 million new cases of genital chlamydial infection occureach year. The Chlamydia of the present invention include, for example,C. trachomatis, Chlamydophila pneumoniae, C. muridarum, C. suis,Chlamydophila abortus, Chlamydophila psittaci, Chlamydophila caviae,Chlamydophila felis, Chlamydophila pecorum, and C. pneumoniae. Animalmodels of chlamydial infection have established that T-cells play acritical role both in the clearance of the initial infection and inprotection from re-infection of susceptible hosts. Hence, theimmunogenic compositions as disclosed herein can be used to provideparticular value by eliciting cellular immune responses againstchlamydial infection.

More specifically, Chlamydial antigens useful in the present inventioninclude DNA gyrase subunit B, sulfite synthesis/biphosphate phosphatase,cell division protein FtsY, methionyl-tRNA synthetase, DNA helicase(uvrD); ATP synthase subunit I (atpI) or a metal-dependent hydrolase(U.S. Patent Application Pub. No. 20090028891). Additional Chlamyidiatrachomatis antigens include CT144 polypeptide, a peptide having aminoacid residues 67-86 of CT144, a peptide having amino acid residues 77-96of CT144, CT242 protein, a peptide having amino acids 109-117 of CT242,a peptide having a mino acids 112-120 of CT242 polypeptide, CT812protein (from the pmpD gene), a peptide having amino acid residues103-111 of the CT812 protein; and several other antigenic peptides fromC. trachomatis: NVTQDLTSSTAKLECTQDLI (SEQ ID NO: 29),AKLECTQDLIAQGKLIVTNP (SEQ ID NO: 30), SNLKRMQKI (SEQ ID NO: 31),AALYSTEDL (SEQ ID NO: 32), FQEKDADTL (SEQ ID NO: 33), QSVNELVYV (SEQ IDNO: 34), LEFASCSSL (SEQ ID NO: 35), SQAEGQYRL (SEQ ID NO: 36), GQSVNELVY(SEQ ID NO: 37), and QAVLLLDQI (SEQ ID NO: 38). See WO 2009/020553.

Additionally, Chlamydia pneumoniae antigens including homologues of theforegoing polypeptides (see U.S. Pat. No. 6,919,187), can be used anantigens in the immunogenic compositions and methods as disclosedherein.

Fungal antigens can be derived from Candida species and other yeast; orother fungi (aspergillus, other environmental fungi). Regarding otherparasites, malaria as well as worms and amoebae may provide theantigenic antigen for use in the in the immunogenic compositions andmethods as disclosed herein.

In some embodiments, where the antigen is to generate an anti-influenzaimmunogen, the surface glycoproteins hemagglutinin (HA) andneuraminidase (NA) are generally the antigens of choice. Bothnucleoprotein (NP) polypeptide and matrix (M) are internal viralproteins and therefore not usually considered in vaccine design forantibody-based immunity. Influenza vaccines are used routinely inhumans, and include vaccines derived from inactivated whole influenzavirus, live attenuated influenza virus, or purified and inactivatedmaterials from viral strains. For example, a traditional influenzavaccine can be manufactured using three potentially threatening strainsof flu virus. These strains are usually grown in fertilized chickeneggs, which requires extensive processing including egg inoculation andincubation, egg harvest, virus purification and inactivation, processingand pooling the virus or viral components to the final vaccineformulation, and aseptic filling in the appropriate containers.Typically, this egg-based production cycle takes over 70 weeks. In theevent of a major influenza epidemic, the availability of a potent andsafe vaccine is a major concern. Additionally, there are risksassociated with impurities in eggs, such as antibiotics andcontaminants, that negatively impact vaccine sterility. Moreover,egg-derived flu vaccines are contraindicated for those with severeallergies to egg proteins and people with a history of Guillain-Barrésyndrome. The present invention provides an alternative to the egg-basedinfluenza vaccines, not only be avoiding egg-related selequae, but beproviding a platform for the use of multiple influenza antigens in ahighly controlled platform.

In some embodiments, an antigen for use in the immunogenic compositionsas disclosed herein can also include those used in biological warfare,such as ricin, which may provoke a CMI response.

Additionally, the present invention also provides immunogeniccompositions comprising antigens which raise an immune response againstcancer. In these conjugates, an antigen is an antigen expressed by acancer or tumor, or derived from a tumor. In some embodiments, suchantigens are referred to herein as a “cancer antigen” and are typicallya protein expressed predominantly on the cancer cells, such that theconjugate elicits both potent humoral and potent cellular immunity tothis protein. A large number of cancer-associated antigens have beenidentified, several of which are now being used to make experimentalcancer treatment vaccines and are thus suitable for use in the presentembodiments. Antigens associated with more than one type of cancerinclude Carcinoembryonic antigen (CEA); Cancer/testis antigens, such asNY-ESO-1; Mucin-1 (MUC1) such as Sialyl Tn (STn); Gangliosides, such asGM3 and GD2; p53 protein; and HER2/neu protein (also known as ERBB2).Antigens unique to a specific type of cancer include a mutant form ofthe epidermal growth factor receptor, called EGFRvIII;Melanocyte/melanoma differentiation antigens, such as tyrosinase, MART1,gp100, the lineage related cancer-testis group (MAGE) andtyrosinase-related antigens; Prostate-specific antigen;Leukaemia-associated antigens (LAAs), such as the fusion proteinBCR-ABL, Wilms' tumour protein and proteinase 3; and Idiotype (Id)antibodies. See, e.g., Mitchell, 3 Curr. Opin. Investig. Drugs 150(2002); Dao & Scheinberg, 21 Best Pract. Res. Clin. Haematol. 391(2008).

Another approach in generating an immune response against cancer employsantigens from microbes that cause or contribute to the development ofcancer. These vaccines have been used against cancers includinghepatocellular carcinoma (hepatitis B virus, hepatitis C virus,Opisthorchis viverrin), lymphoma and nasoparyngeal carcinoma(Epstei-Barr virus), colorectal cancer, stomach cancer (Helicobacterpylori), bladder cancer (Schisosoma hematobium), T-cell leukemia (humanT-cell lymphtropic virus), cervical cancer (human papillomavirus), andothers. To date, there have been clinical trials for vaccines targetingBladder Cancer, Brain Tumors, Breast Cancer, Cervical Cancer, KidneyCancer, Melanoma, Multiple Myeloma, Leukemia, Lung Cancer, PancreaticCancer, Prostate Cancer, and Solid Tumors. See Pardoll et al., ABELOFF'SCLIN. ONCOL. (4th ed., Churchill Livingstone, Philadelphia 2008); Sioud,360 Methods Mol. Bio. 277 (2007); Pazdur et al., 30 J. Infusion Nursing30(3):173 (2007); Parmiani et al., 178 J. Immunol. 1975 (2007); Lolliniet al., 24 Trends Immunol. 62 (2003); Schlom et al., 13 Clin. CancerRes. 3776 (2007); Banchereau et al., 392 Nature 245 (1998); Finn, 358New Engl. J. Med. 2704 (2008); Curigliano et al., 7 Exp. Rev. AnticancerTher. 1225 (2007). Marek's Disease virus, a herpes virus that causestumors in poultry, has long been managed by vaccine. Thus, the presentembodiments encompass both preventive or prophylactic anti-cancerimmunogenic compositions and treatment/therapeutic cancer vaccines.

Contemplated proliferative diseases and cancers include AIDS relatedcancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloidleukemia, adenocystic carcinoma, adrenocortical cancer, agnogenicmyeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer,angiosarcoma, astrocytoma, ataxia-telangiectasia, basal cell carcinoma(skin), bladder cancer, bone cancers, bowel cancer, brain and CNStumors, breast cancer, carcinoid tumors, cervical cancer, childhoodbrain tumours, childhood cancer, childhood leukemia, childhood softtissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocyticleukemia, chronic myeloid leukemia, colorectal cancers, cutaneous t-celllymphoma, dermatofibrosarcoma-protuberans,desmoplastic-small-round-cell-tumour, ductal carcinoma, endocrinecancers, endometrial cancer, ependymoma, esophageal cancer, Ewing'ssarcoma, extra-hepatic bile duct cancer, eye cancer, including, e.g.,eye melanoma and retinoblastoma, fallopian tube cancer, fanconi anemia,fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinalcancers, gastrointestinal-carcinoid-tumour, genitourinary cancers, germcell tumors, gestational-trophoblastic disease, glioma, gynecologicalcancers, hematological malignancies, hairy cell leukemia, head and neckcancer, hepatocellular cancer, hereditary breast cancer, Hodgkin'sdisease, human papillomavirus-related cervical cancer, hydatidiformmole, hypopharynx cancer, islet cell cancer, Kaposi's sarcoma, kidneycancer, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumenisyndrome, lip cancer, liposarcoma, lung cancer, lymphedema, lymphoma,non-Hodgkin's lymphoma, male breast cancer,malignant-rhabdoid-tumour-of-kidney, medulloblastoma, melanoma, Merkelcell cancer, mesothelioma, metastatic cancer, mouth cancer, multipleendocrine neoplasia, mycosis fungoides, myelodysplastic syndromes,myeloma, myeloproliferative disorders, nasal cancer, nasopharyngealcancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegenbreakage syndrome, non-melanoma skin cancer,non-small-cell-lung-cancer-(NSCLC), oral cavity cancer, oropharynxcancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasalcancer, parathyroid cancer, parotid gland cancer, penile cancer,peripheral-neuroectodermal-tumours, pituitary cancer, polycythemia vera,prostate cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma,Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, Schwannoma,Sezary syndrome, skin cancer, small cell lung cancer (SCLC), smallintestine cancer, soft tissue sarcoma, spinal cord tumours,squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma,testicular cancer, thymus cancer, thyroid cancer,transitional-cell-cancer-(bladder), transitional-cell-cancer(renal-pelvis/ureter), trophoblastic cancer, urethral cancer, urinarysystem cancer, uterine sarcoma, uterus cancer, vaginal cancer, vulvacancer, Waldenstrom's-macroglobulinemia, and Wilms' tumor.

In some embodiments, an antigen for use in the immunogenic compositionsas disclosed herein can include antigens of autoimmune diseases, e.g.,they can be “self-antigens.” Autoimmune diseases contemplated fordiagnosis according to the assays described herein include, but are notlimited to alopecia areata, ankylosing spondylitis, antiphospholipidsyndrome, Addison's disease, aplastic anemia, multiple sclerosis,autoimmune disease of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, Behcet'sDisease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis,chronic fatigue syndrome, chronic inflammatory demyelinating syndrome(CFIDS), chronic inflammatory polyneuropathy, Churg-Strauss syndrome,cicatricial pemphigoid, CREST Syndrome, cold agglutinin disease, Crohn'sdisease, dermatitis herpetiformis, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia, glomerulonephritis, Grave's disease,Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulindependent diabetes (Type I), Lichen Planus, lupus, Meniere's Disease,mixed connective tissue disease, myasthenia gravis, myocarditis,pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome,rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma,Sjogren's syndrome, stiff-man syndrome, Takayasu arteritis, temporalarteritis/giant cell arteritis, ulcerative colitis, uveitis, Wegener'ssyndrome, vasculitis and vitiligo. It is generally important to assessthe potential or actual CMI responsiveness in subjects having, orsuspected of having or being susceptible to an autoimmune disease.

In some embodiments, an antigen for use in the immunogenic compositionsas disclosed herein can be an antigen which is associated with aninflammatory disease or condition. Examples of inflammatory diseaseconditions where antigens may be useful include but are not limited toacne, angina, arthritis, aspiration pneumonia, empyema, gastroenteritis,necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis,pleurisy, chronic inflammatory demyelinating polyneuropathy, chronicinflammatory demyelinating polyradiculoneuropathy, and chronicinflammatory demyelinating polyneuropathy, among others.

In some embodiments, an antigen can be an intact (i.e., an entire orwhole) antigen, or a functional portion of an antigen that comprisesmore than one epitope. In some embodiments, an antigen is a peptidefunctional portion of an antigen. By “intact” in this context is meantthat the antigen is the full length antigen as that antigen polypeptideoccurs in nature. This is in direct contrast to delivery of only a smallportion or peptide of the antigen. Delivering an intact antigen to acell enables or facilitates eliciting an immune response to a full rangeof epitopes of the intact antigen, rather than just a single or selectedfew peptide epitopes. Accordingly, the methods and immunogeniccompositions described herein encompass intact antigens associated withthe polymer for a more sensitive and have higher specificity of immuneresponse as compared to use of a single epitope peptide-based antigen.

Alternatively, in some embodiments, an intact antigen can be dividedinto many parts, depending on the size of the initial antigen.Typically, where a whole antigen is a multimer polypeptide, the wholeprotein can be divided into sub-units and/or domains where eachindividual sub-unit or domain of the antigen can be associated with thepolymer according to the methods as disclosed herein. Alternatively, insome embodiments, an intact antigen can be divided into functionalfragments, or parts, of the whole antigen, for example, at least two, orat least 3, or at least 4, or at least 5, or at least 6, or at least 7,or at least 8, or at least 9, or at least 10, or at least 11, or atleast 12, or at least 13, or at least 15, or at least 20, or at least25, or more than 25 portions (e.g., pieces or fragments), inclusive, andwhere each individual functional fragment of the antigen can beassociated with the polymer according to the methods as disclosedherein.

The fragmentation or division of a full length antigen polypeptide canbe an equal division of the full length antigen polypeptide, oralternatively, in some embodiments, the fragmentation is asymmetrical orunequal. As a non-limiting example, where an antigen is divided into twooverlapping fragments, an antigen can be divided into fragments ofapproximately the same (equal) size, or alternatively one fragment canbe about 45% of the whole antigen and the other fragment can be about65%. As further non-limiting examples, a whole antigen can be dividedinto a combination of differently sized fragments, for example, where anantigen is divided into two fragments, fragments can be divided intoabout 40% and about 70%, or about 45% and about 65%; or about 35% andabout 75%; or about 25% and about 85%, inclusive, of the whole antigen.Any combination of overlapping fragments of a full length whole antigenis encompassed for use in the generation of a panel of overlappingpolypeptides of an antigen. As an illustrative example only, where aantigen is divided into 5 portions, the portions can divided equally(i.e., each overlapping fragment is about 21% to 25% of the entire fulllength if the antigen) or unequally (i.e., an antigen can be dividedinto the following five overlapping fragments; fragment 1 is about 25%,fragment 2 is about 5%, fragment 3 is about 35%, fragment 4 is about 10%and fragment 5 is about 25% of the size of the full length antigen,provided each fragment overlaps with at least one other fragment).

Typically, a panel of antigen portions can substantially cover theentire length of the whole (or intact) antigen polypeptide. Accordingly,in some embodiments, an immunogenic composition comprises a polymer withmany different, and/or overlapping fragments of the same intact antigen.Overlapping protein fragments of a antigen can be produced much quickerand cheaper, and with increased stability as compared to the use ofpeptide antigens alone. Further in some embodiments, antigens which arepolypeptides larger than simple peptides are preferred as conformationis important for epitope recognition, and the larger antigenpolypeptides or fragments will provide a benefit over peptide fragments.

One of ordinary skill in the art can divide a whole antigen intooverlapping proteins of an antigen to create a panel of polypeptides ofthe antigen. By way of an illustrative example only, the TB-specificantigen TB1 (CFP also known as culture filtrate-10 or CFP-10) can bedivided into, for example at least seventeen portions to generate apanel of seventeen different polypeptides, each comprising a differentbut overlapping TB-specific antigen TB1 (CFP) fragment. Culture filtrateprotein (CFP-10) (Genbank AAC83445) is a 10 kDa, 100 amino acid residueprotein fragment from M. tuberculosis. It is also known as L45 antigenhomologous protein (LHP).

A target antigen for use in the methods and compositions describedherein can be expressed by recombinant means, and can optionally includean affinity or epitope tag to facilitate purification, which methods arewell-known in the art. Chemical synthesis of an oligopeptide, eitherfree or conjugated to carrier proteins, can be used to obtain antigen ofthe invention. Oligopeptides are considered a type of polypeptide. Anantigen can be expressed as a fusion with a complementary affinitymolecule, e.g., but not limited to rhizavidin or a derivative orfunctional fragment thereof. Alternatively, it is also possible toprepare target antigen and then conjugate it to a complementary affinitymolecule, e.g., but not limited to rhizavidin or a derivative orfunctional fragment thereof.

Polypeptides can also by synthesized as branched structures such asthose disclosed in U.S. Pat. Nos. 5,229,490 and 5,390,111. Antigenicpolypeptides include, for example, synthetic or recombinant B-cell andT-cell epitopes, universal T-cell epitopes, and mixed T-cell epitopesfrom one organism or disease and B-cell epitopes from another.

An antigen can obtained through recombinant means or chemicalpolypeptide synthesis, as well as antigen obtained from natural sourcesor extracts, can be purified by means of the antigen's physical andchemical characteristics, such as by fractionation or chromatography.These techniques are well-known in the art.

In some embodiments, an antigen can be solubilized in water, a solventsuch as methanol, or a buffer. Suitable buffers include, but are notlimited to, phosphate buffered saline Ca²⁺/Mg²⁺ free (PBS), normalsaline (150 mM NaCl in water), and Tris buffer. Antigen not soluble inneutral buffer can be solubilized in 10 mM acetic acid and then dilutedto the desired volume with a neutral buffer such as PBS. In the case ofantigen soluble only at acid pH, acetate-PBS at acid pH can be used as adiluent after solubilization in dilute acetic acid. Glycerol can be asuitable non-aqueous solvent for use the compositions, methods and kitsdescribed herein.

Typically, when designing a protein vaccine against a pathogen, anextracellular protein or one exposed to the environment on a virus isoften the ideal candidate as the antigen component in the vaccine.Antibodies generated against that extracellular protein become the firstline of defense against the pathogen during infection. The antibodiesbind to the protein on the pathogen to facilitate antibody opsonizationand mark the pathogen for ingestion and destruction by a phagocyte suchas a macrophage. Antibody opsonization can also kill the pathogen byantibody-dependent cellular cytotoxicity. The antibody triggers arelease of lysis products from cells such as monocytes, neutrophils,eosinophils, and natural killer cells.

In one embodiment of the invention described herein, antigens for use inthe compositions as disclosed herein all wild type proteins, as in theamino acid residues have the sequences found in naturally occurringviruses and have not been altered by selective growth conditions ormolecular biological methods.

In one embodiment, the immunogenic compositions described as herein cancomprise antigens which are glycosylated proteins. In other words, anantigen of interest can each be a glycosylated protein. In oneembodiment of the immunogenic compositions as described herein,antigens, or antigen-fusion polypeptides are O-linked glycosylated. Inanother embodiment of the immunogenic compositions as described herein,antigens, or antigen-fusion polypeptides are N-linked glycosylated. Inyet another embodiment of the immunogenic compositions as describedherein, antigens, or antigen-fusion are both O-linked and N-linkedglycosylated. In other embodiments, other types of glycosylations arepossible, e.g., C-mannosylation. Glycosylation of proteins occurspredominantly in eukaryotic cells. N-glycosylation is important for thefolding of some eukaryotic proteins, providing a co-translational andpost-translational modification mechanism that modulates the structureand function of membrane and secreted proteins. Glycosylation is theenzymatic process that links saccharides to produce glycans, andattaches them to proteins and lipids. In N-glycosylation, glycans areattached to the amide nitrogen of asparagine side chain during proteintranslation. The three major saccharides forming glycans are glucose,mannose, and N-acetylglucosamine molecules. The N-glycosylationconsensus is Asn-Xaa-Ser/Thr, where Xaa can be any of the known aminoacids. O-linked glycosylation occurs at a later stage during proteinprocessing, probably in the Golgi apparatus. In O-linked glycosylation,N-acetyl-galactosamine, 0-fucose, 0-glucose, and/or N-acetylglucosamineis added to serine or threonine residues. One skilled in the art can usebioinformatics software such as NetNGlyc 1.0 and NetOGlyc Predictionsoftwares from the Technical University of Denmark to find the N- andO-glycosylation sites in a polypeptide in the present invention. TheNetNglyc server predicts N-Glycosylation sites in proteins usingartificial neural networks that examine the sequence context ofAsn-Xaa-Ser/Thr sequons. The NetNGlyc 1.0 and NetOGlyc 3.1 Predictionsoftware can be accessed at the EXPASY website. In one embodiment,N-glycosylation occurs in the target antigen polypeptide of the fusionpolypeptide described herein.

Affinity Molecule Pairs

As disclosed herein, in some embodiments, an antigen is connected to apolymer via complementary affinity pairs. This connecting of the antigento the polymer is mediated by the polymer being connected to a firstaffinity molecule, which associates a second (e.g., complementary)affinity molecule, which is attached to the antigen. An examplecomplementary affinity pair is biotin/biotin-binding protein.

Exemplary examples of the affinity complementary affinity pairs include,but without limitation, biotin-binding proteins or avidin-like proteinsthat bind to biotin. For example, where the first affinity bindingmolecule is biotin (which associates with the polymer), thecomplementary affinity molecule can be a biotin-binding protein or anavidin-like protein or a derivative thereof, e.g., but not limited to,avidin, rhizavidin, or streptavidin or variants, derivatives orfunctional portions thereof.

In some embodiments, the first affinity binding molecule is biotin, abiotin derivative, or a biotin mimic, for example, but not limited to,amine-PEG3-biotin (((+)-biotinylation-3-6,9-trixaundecanediamine) or aderivative or functional fragment thereof. A specific biotin mimetic hasa specific peptide motif containing sequence of DX_(a)AX_(b)PX_(c) (SEQID NO: 39), or CDX_(a)AX_(b)PX_(c)CG (SEQ ID NO: 40), where X_(a) is Ror L, X_(b) is S or T, and X_(c) is Y or W. These motifs can bind avidinand Neutravidin, but streptavidin. See, e.g., Gaj et al., 56 Prot.Express. Purif. 54 (2006).

The linkage of the first affinity molecule to the polymer, and thecomplementary affinity molecule to the antigen can be a non-covalentlinkage, or a chemical mechanism, for instance covalent binding,affinity binding, intercalation, coordinate binding and complexation.Covalent binding provides for very stable binding, and is particularlywell-suited for the present embodiments. Covalent binding can beachieved either by direct condensation of existing side chains or by theincorporation of external bridging molecules.

For example, in some embodiments, an antigen can be non-covalentlybonded to one of the pairs in a complementary affixing pair. Inalternative embodiments, an antigen can be covalently bonded or fused toone of the pairs in a complementary affixing pair. Methods forgeneration of fusion proteins are well known in the art, and arediscussed herein.

In other embodiments, a first affinity binding molecule is linked to thepolymer by a non-covalent bond, or by a covalent bond. In someembodiments, a cross-linking reagent is used to covalently bond thefirst affinity binding molecule to the polymer as disclosed herein.

In some embodiments, the first affinity binding molecule associates withthe complementary affinity molecule by non-covalent bond association asknown in the art, including, but not limited to, electrostaticinteraction, hydrogen bound, hydrophobic interaction (i.e., van derWaals forces), hydrophilic interactions, and other non-covalentinteractions. Other higher order interactions with intermediate moietiesare also contemplated.

In some embodiments, the complementary affinity molecule is anavidin-related polypeptide. In specific embodiments, the complementaryaffinity molecule is rhizavidin, such as recombinant rhizavidin. Inparticular, the recombinant rhizavidin is a modified rhizavidin that canbe expressed in E. coli with a high yield. The typical yield is >30 mgper liter of E. coli culture. Rhizavidin has a lower sequence homologyto egg avidin (22.4% sequence identity and 35.0% similarity) comparedwith other avidin-like proteins. Use of the modified rhizavidin reducesthe risk of the MAPS inducing an egg-related allergic reaction in asubject. Moreover, antibody to recombinant modified rhizavidin has noapparent cross-reactivity to egg avidin (and vice versa).

More specifically, some embodiments comprise a modified rhizavidindesigned for recombinant expression in E. coli. The coding sequence forthe rhizavidin gene was optimized using E. coli expression codons, toavoid any difficulty during expression in E. coli due to rare codonspresent in original gene. To simplify the construct, after abioinformatics and structure-based analysis, the first 44 residues offull length rhizavidin were removed, as these were found to beunnecessary for the core structure and function. The correct folding ofrecombinant protein was improved by added an E. coli secretion signalsequence to the N-terminal of the shortened rhizavidin (45-179), tofacilitate the translocation of recombinant protein into the periplasmicspace of E. coli cells where the functionally important disulfide bondin rhizavidin can form correctly. The modified recombinant rhizavidinforms a dimer, compared with known avidin-like proteins which formtetramers, further improving expression of the recombinantrhizavidin-antigen fusion as a soluble protein in E. coli.

Moreover, as discussed in further detail elsewhere herein, to improvethe expression and solubility of fusion antigens in E. coli, a flexiblelinker region was added between rhizavidin and the antigen protein.Additionally, based on the biotinformatics and structural analysis,different antigen constructs were cloned and expressed: either fulllength antigen, or the important functional domain, or chimera proteinswere comprising with two different antigens.

Additional affinity pairs that may be useful in the methods andcompositions described herein include antigen-antibody,metal/ion-metal/ion-binding protein, lipid/lipid binding protein,saccharide/saccharide binding protein, amino acid/peptide/amino acid orpeptide binding protein, enzyme-substrate or enzyme-inhibitor,ligand-agonist/receptor, or biotin mimetic. When using alternativeaffinity pairs, alternative means of attaching the respective polymerand antigen may also be employed, such as in vitro enzymatic reactionsrather than genetic fusion. More specifically, antigen-antibody affinitypair provides for a very strong and specific interaction. The antigencan be any epitope including protein, peptide, nucleic acid, lipid,poly/oligosaccharide, ion, etc. The antibody can be any type ofimmunoglobulin, or the Ag-binding portion of an immunoglobulin, such asa Fab fragment. Regarding metal/ion-metal/ion binding protein, examplesinclude Ni NTA vs. histidine-tagged protein, or Zn vs. Zn bindingprotein. Regarding lipid/lipid binding protein, examples includecholesterol vs. cholesterol binding protein. Regardingsaccharide/saccharide binding protein, examples include maltose vs.maltose binding protein, mannose/glucose/oligosaccharide vs. lectin.Enzyme-substrate/inhibitors include substrates from a wide range ofsubstances, including protein, peptide, amino acid, lipid, sugar, orions. The inhibitor can be the analog of the real substrate which cangenerally bind to the enzymes more tightly and even irreversibly. Forexample, trypsin vs. soy trypsin inhibitor. The inhibitor can be naturalor synthetic molecule. Regarding other ligand/agonist-receptor, ligandcan be from a wide range of substance, including protein, peptide, aminoacid, lipid, sugar, ion, agonist can be the analog of the real ligand.Examples include the LPS vs. TLR4 interaction.

Cross-Linking Reagents:

Many bivalent or polyvalent linking agents are useful in couplingprotein molecules to other molecules. For example, representativecoupling agents can include organic compounds such as thioesters,carbodiimides, succinimide esters, disocyanates, glutaraldehydes,diazobenzenes and hexamethylene diamines. This listing is not intendedto be exhaustive of the various classes of coupling agents known in theart but, rather, is exemplary of the more common coupling agents. SeeKillen & Lindstrom, 133 J. Immunol. 1335 (1984); Jansen et al., 62 Imm.Rev. 185 (1982); Vitetta et al.

In some embodiments, cross-linking reagents agents described in theliterature are encompassed for use in the methods, immunogeniccompositions and kits as disclosed herein. See, e.g., Ramakrishnan, etal., 44 Cancer Res. 201 (1984) (describing the use of MBS(M-maleimidobenzoyl-N-hydroxysuccinimide ester)); Umemoto et al., U.S.Pat. No. 5,030,719 (describing the use of a halogenated acetyl hydrazidederivative coupled to an antibody by way of an oligopeptide linker).Particular linkers include: (a) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (b) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (c) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat#21651G); (d) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (f) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkages or linking agents described above contain components thathave different attributes, thus leading to conjugates with differingphysio-chemical properties. For example, sulfo-NHS esters of alkylcarboxylates are more stable than sulfo-NHS esters of aromaticcarboxylates. NHS-ester containing linkers are less soluble thansulfo-NHS esters. Further, the linker SMPT contains a stericallyhindered disulfide bond, and can form conjugates with increasedstability. Disulfide linkages, are in general, less stable than otherlinkages because the disulfide linkage can be cleaved in vitro,resulting in less conjugate available. Sulfo-NHS, in particular, canenhance the stability of carbodimide couplings. Carbodimide couplings(such as EDC) when used in conjunction with sulfo-NHS, forms esters thatare more resistant to hydrolysis than the carbodimide coupling reactionalone.

Exemplary cross-linking molecules for use in the methods and immunogeniccompostions as disclosed herein include, but are not limited to thoselisted in Tables 2 and 3.

TABLE 2 Exemplary homobifunctional crosslinkers* Crosslinker ReactiveCrosslinking Target Groups, Features Example Products Amine-to-Amine NHSesters DSG; DSS; BS3; TSAT (trifunctional); Bioconjugate Toolkit ReagentPairs NHS esters, PEG spacer BS(PEG)5; BS(PEG)9 NHS esters,thiol-cleavable DSP; DTSSP NHS esters, misc-cleavable DST; BSOCOES; EGS;Sulfo-EGS Imidoesters DMA; DMP; DMS Imidoesters, thiol-cleavable DTBPOther DFDNB; THPP (trifunctional); Aldehyde-Activated Dextran KitSulfhydryl-to-Sulfhydryl Maleimides BMOE; BMB; BMH; TMEA (trifunctional)Maleimides, PEG spacer BM(PEG)2; BM(PEG)3 Maleimides, cleavable BMDB;DTME Pyridyldithiols (cleavable) DPDPB Other HBVS (vinylsulfone)Nonselective Aryl azides BASED (thiol-cleavable) *crosslinking reagentsthat have the same type of reactive group at either end. Reagents areclassified by what chemical groups they cross link (left column) andtheir chemical composition (middle column). Products are listed in orderof increasing length within each cell.

TABLE 3 Exemplary heterobifunctional crosslinkers* Crosslinker ReactiveCrosslinking Targets Groups, Features Example ProductsAmine-to-Sulfhydryl NHS ester/Maleimide AMAS; BMPS; GMBS and Sulfo-GMBS; MBS and Sulfo-MBS; SMCC and Sulfo-SMCC; EMCS and Sulfo- EMCS; SMPBand Sulfo-SMPB; SMPH; LC-SMCC; Sulfo-KMUS NHS ester/Maleimide, SM(PEG)2;SM(PEG)4; SM(PEG)6; PEG spacer SM(PEG)8; SM(PEG)12; SM(PEG)24 NHSester/Pyridyldithiol, SPDP; LC-SPDP and Sulfo-LC-SPDP; cleavable SMPT;Sulfo-LC-SMPT NHS esters/Haloacetyl SIA; SBAP; SIAB; Sulfo-SIABAmine-to-Nonselective NHS ester/Aryl Azide NHS-ASA ANB-NOS Sulfo-HSABSulfo-NHS-LC-ASA SANPAH and Sulfo-SANPAH NHS ester/Aryl Azide,Sulfo-SFAD; Sulfo-SAND; Sulfo- cleavable SAED NHS ester/Diazirine SDAand Sulfo-SDA; LC-SDA and Sulfo-LC-SDA NHS ester/Diazirine, SDAD andSulfo-SDAD cleavable Amine-to-Carboxyl Carbodiimide DCC; EDCSulfhydryl-to-Nonselective Pyridyldithiol/Aryl Azide APDPSulfhydryl-to-Carbohydrate Maleimide/Hydrazide BMPH; EMCH; MPBH; KMUHPyridyldithiol/Hydrazide BMPH; EMCH; MPBH; KMUHCarbohydrate-to-Nonselective Hydrazide/Aryl Azide ABHHydroxyl-to-Sulfhydryl Isocyanate/Maleimide PMPI Amine-to-DNA NHSester/Psoralen SPB *crosslinking reagents that have the differentreactive groups at either end. Reagents are classified by what chemicalgroups they cross link (left column) and their chemical composition(middle column). Products are listed in order of increasing lengthwithin each cell.Co-Stimulatory Factor

In some embodiments, the immunogenic composition as disclosed hereincomprises at least one co-stimulatory molecule. In some embodiments, theco-stimulatory factor is cross-linked to the polymer. In someembodiments, the co-stimulatory factor is associated to the polymer by acomplementary affinity pair similar to as an antigen is associated withthe polymer. In some embodiments, where the complementary affinity pairwhich links the co-stimulatory factor to the polymer is the same, or adifferent complementary affinity pair which links the antigen to thepolymer.

In some embodiments, at least one, or at least 2, or at least 3, or atleast 5, or at least 10, or at least 15, or at least 20, or at least 50,or at least 100, or more than about 100, inclusive, co-stimulatoryfactors can be associated with the polymer as disclosed herein. In someembodiments, the co-stimulatory factors can be the same co-stimulatorfactor, or they can be a variety of different co-stimulatory factorsassociated with the polymer.

In some embodiments, the co-stimulator factor is a ligand/agonist ofToll like receptors, e.g., but not limited to TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, etc. In some embodiments, aco-stimulator factor is a NOD ligand/agonist, or an activator/agonist ofthe inflammasome. Without wishing to be bound by theory, theinflammasome is a multiprotein oligomer consisting of caspase 1, PYCARD,NALP and sometimes caspase 5 or caspase 11 and promotes the maturationof inflammatory cytokines interleukin 1-13 and interleukin 18.

In some embodiments, a co-stimulator factor is a cytokine. In someembodiments, a cytokine is selected from the group consisting of:GM-CSF; IL-1α; IL-1β; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10;IL-12; IL-23; IFN-α; IFN-β; IFN-β; IFN-γ; MIP-1α; MIP-1β; TGF-β; TNFα,and TNFβ. In some embodiments, the co-stimulatory factor is an adjuvant,which may be associated with the polymer, as just discussed, or may beadded to the MAPS composition prior to or concurrent with administrationto a subject. Adjuvants are further described elsewhere herein.

Production of Recombinant Proteins

Recombinant proteins may be conveniently expressed and purified by aperson skilled in the art, or by using commercially available kits, forexample PRoBoND™ Purification System (Invitrogen Corp., Carlsbad,Calif.). In some embodiments, recombinant antigens can be synthesizedand purified by protein purification methods using bacterial expressionsystems, yeast expression systems, baculovirus/insect cell expressionsystem, mammalian cell expression systems, or transgenic plant or animalsystems as known to persons of ordinary skill in the art.

The proteins, polypeptides and fusion polypeptides described herein canall be synthesized and purified by protein and molecular methods thatare well known to one skilled in the art. Molecular biology methods andrecombinant heterologous protein expression systems are used. Forexample, recombinant protein can be expressed in bacteria, mammalian,insect, yeast, or plant cells; or in transgenic plant or animal hosts.

In one embodiment, provided herein is an isolated polynucleotideencoding a fusion polypeptide or a non-fusion polypeptide describedherein. Conventional polymerase chain reaction (PCR) cloning techniquescan be used to construct a chimeric or fusion coding sequence encoding afusion polypeptide as described herein. A coding sequence can be clonedinto a general purpose cloning vector such as pUC19, pBR322,pBLUESCRIPT® vectors (Stratagene, Inc.) or pCR TOPO® (Invitrogen). Theresultant recombinant vector carrying the nucleic acid encoding apolypeptide as described herein can then be used for further molecularbiological manipulations such as site-directed mutagenesis to create avariant fusion polypeptide as described herein or can be subcloned intoprotein expression vectors or viral vectors for protein synthesis in avariety of protein expression systems using host cells selected from thegroup consisting of mammalian cell lines, insect cell lines, yeast,bacteria, and plant cells.

Each PCR primer should have at least 15 nucleotides overlapping with itscorresponding templates at the region to be amplified. The polymeraseused in the PCR amplification should have high fidelity such asPfuULTRA® polymerase (Stratagene) for reducing sequence mistakes duringthe PCR amplification process. For ease of ligating several separate PCRfragments together, for example in the construction of a fusionpolypeptide, and subsequently inserting into a cloning vector, the PCRprimers should also have distinct and unique restriction digestion siteson their flanking ends that do not anneal to the DNA template during PCRamplification. The choice of the restriction digestion sites for eachpair of specific primers should be such that the fusion polypeptidecoding DNA sequence is in-frame and will encode the fusion polypeptidefrom beginning to end with no stop codons. At the same time the chosenrestriction digestion sites should not be found within the coding DNAsequence for the fusion polypeptide. The coding DNA sequence for theintended polypeptide can be ligated into cloning vector pBR322 or one ofits derivatives, for amplification, verification of fidelity andauthenticity of the chimeric coding sequence, substitutions/or specificsite-directed mutagenesis for specific amino acid mutations andsubstitutions in the polypeptide.

Alternatively the coding DNA sequence for the polypeptide can be PCRcloned into a vector using for example, the TOPO® cloning methodcomprising topoisomerase-assisted TA vectors such as pCR®-TOPO,pCR®-Blunt II-TOPO, pENTR/D-TOPO®, and pENTR/SD/D-TOPO®. (Invitrogen,Inc., Carlsbad, Calif.). Both pENTR/D-TOPO®, and pENTR/SD/D-TOPO® aredirectional TOPO entry vectors which allow the cloning of the DNAsequence in the 5′→3′ orientation into a GATEWAY® expression vector.Directional cloning in the 5′→3′ orientation facilitates theunidirectional insertion of the DNA sequence into a protein expressionvector such that the promoter is upstream of the 5′ ATG start codon ofthe fusion polypeptide coding DNA sequence, enabling promoter drivenprotein expression. The recombinant vector carrying the coding DNAsequence for the fusion polypeptide can be transfected into andpropagated in general cloning E. coli such as XL1Blue, SURE®(STRATAGENE®) and TOP-10 cells (Invitrogen).

One skilled in the art would be able to clone and ligate the codingregion of the antigen of interest with the coding region of thecomplementary affinity molecule to construct a chimeric coding sequencefor a fusion polypeptide comprising the antigen or a fragment thereofand the complementary affinity molecule of a derivative thereof usingspecially designed oligonucleotide probes and polymerase chain reaction(PCR) methodologies that are well known in the art. One skilled in theart would also be able to clone and ligate the chimeric coding sequencefor a fusion protein into a selected vector, e.g., bacterial expressionvector, an insect expression vector or baculovirus expression vector.The coding sequences of antigen and the target antigen polypeptide orfragment thereof should be ligated in-frame and the chimeric codingsequence should be ligated downstream of the promoter, and between thepromoter and the transcription terminator. Subsequent to that, therecombinant vector is transfected into regular cloning E. coli, such asXL1Blue. Recombinant E. coli harboring the transfer vector DNA is thenselected by antibiotic resistance to remove any E. coli harboringnon-recombinant plasmid DNA. The selected transformant E. coli are grownand the recombinant vector DNA can be subsequently purified fortransfection into S. frupperda cells.

In some embodiments, the antigens as disclosed herein can comprise asignal peptide for translocation into periplasmic space of bacteria. Thesignal peptide is also called a leader peptide in the N-terminus, whichmay or may not be cleaved off after the translocation through themembrane. One example of a signal peptide is MKKIWLALAGLVLAFSASA (SEQ IDNO: 2) as disclosed herein. Another signal sequence isMAPFEPLASGILLLLWLIAPSRA (SEQ ID NO: 7). Other examples of signalpeptides can be found at SPdb, a Signal Peptide Database, which is foundat the world wide web site of “proline.bic.nus.edu.sg/spdb/”.

In some embodiments, where the antigen is fused to a biotin-bindingprotein, the signal sequence can be located at the N-terminal of thebiotin-binding protein. In some embodiments, the signal sequence iscleaved off from the biotin-binding protein after translocation into theperiplasmic space of E. coli.

In some embodiments, where the antigen is fused to a complementaryaffinity protein, the signal sequence can be located at the N-terminalof the complementary affinity protein. For example, if an antigen isfused to an avidin-like protein, the signal sequence can be located atthe N-terminal of the complementary affinity protein. In someembodiments, the signal sequence is cleaved off from the complementaryaffinity protein before the complementary affinity protein associateswith the first affinity molecule.

In some embodiments, an antigen and/or complementary affinity protein asdescribed herein lacks a signal sequence.

The polypeptides described herein can be expressed in a variety ofexpression host cells e.g., bacteria, yeasts, mammalian cells, insectcells, plant cells, algal cells such as Chlamadomonas, or in cell-freeexpression systems. In some embodiments the nucleic acid can besubcloned from the cloning vector into a recombinant expression vectorthat is appropriate for the expression of fusion polypeptide inbacteria, mammalian, insect, yeast, or plant cells or a cell-freeexpression system such as a rabbit reticulocyte expression system. Somevectors are designed to transfer coding nucleic acid for expression inmammalian cells, insect cells and year in one single recombinationreaction. For example, some of the GATEWAY® (Invitrogen) destinationvectors are designed for the construction of baculovirus, adenovirus,adeno-associated virus (AAV), retrovirus, and lentiviruses, which uponinfecting their respective host cells, permit heterologous expression offusion polypeptides in the appropriate host cells. Transferring a geneinto a destination vector is accomplished in just two steps according tomanufacturer's instructions. There are GATEWAY® expression vectors forprotein expression in insect cells, mammalian cells, and yeast.Following transformation and selection in E. coli, the expression vectoris ready to be used for expression in the appropriate host.

Examples of other expression vectors and host cells are the strong CMVpromoter-based pcDNA3.1 (Invitrogen) and pCINEO vectors (Promega) forexpression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat,and MCF-7; replication incompetent adenoviral vector vectors pADENO-XTM,pAd5F35, pLP-ADENO™-X-CMV (CLONTECH®), pAd/CMV/V5-DEST, pAd-DEST vector(Invitrogen) for adenovirus-mediated gene transfer and expression inmammalian cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for usewith the RETRO-X™ system from Clontech for retroviral-mediated genetransfer and expression in mammalian cells; pLenti4/V5-DEST™,pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen) forlentivirus-mediated gene transfer and expression in mammalian cells;adenovirus-associated virus expression vectors such as pAAV-MCS,pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) for adeno-associatedvirus-mediated gene transfer and expression in mammalian cells; BACpak6baculovirus (Clontech) and pFASTBAC™ HT (Invitrogen) for the expressionin S. frugiperda 9 (Sf9), Sf11, Tn-368 and BTI-TN-5B4-1 insect celllines; pMT/BiP/V5-His (Invitrogen) for the expression in Drosophilaschneider S2 cells; Pichia expression vectors pPICZα, pPICZ, pFLDα andpFLD (Invitrogen) for expression in P. pastoris and vectors pMETα andpMET for expression in P. methanolica; pYES2/GS and pYD1 (Invitrogen)vectors for expression in yeast S. cerevisiae.

Recent advances in the large scale expression heterologous proteins inChlamydomonas reinhardtii are described. Griesbeck., 34 Mol. Biotechnol.213 (2006); Fuhrmann, 94 Methods Mol Med. 191 (2006). Foreignheterologous coding sequences are inserted into the genome of thenucleus, chloroplast and mitochondria by homologous recombination. Thechloroplast expression vector p64 carrying the most versatilechloroplast selectable marker aminoglycoside adenyl transferase (aadA),which confer resistance to spectinomycin or streptomycin, can be used toexpress foreign protein in the chloroplast. The biolistic gene gunmethod can be used to introduce the vector in the algae. Upon its entryinto chloroplasts, the foreign DNA is released from the gene gunparticles and integrates into the chloroplast genome through homologousrecombination.

Also included in the invention are complementary affinity molecule fusedto an antigen. In some embodiments, the fusion construct can alsooptionally comprise purification tags, and/or secretion signal peptides.These fusion proteins may be produced by any standard method. Forexample, for production of a stable cell line expressing anantigen-complementary affinity molecule fusion protein, PCR-amplifiedantigen nucleic acids may be cloned into the restriction site of aderivative of a mammalian expression vector. For example, KA, which is aderivative of pcDNA3 (Invitrogen) contains a DNA fragment encoding aninfluenza virus hemagglutinin tag (HA). Alternatively, vectorderivatives encoding other tags, such as c-myc or poly Histidine tags,can be used. The antigen-complementary affinity molecule fusionexpression construct may be co-transfected, with a marker plasmid, intoan appropriate mammalian cell line (e.g., COS, HEK293T, or NIH 3T3cells) using, for example, LIPOFECTAMINE™ (Gibco-BRL, Gaithersburg, Md.)according to the manufacturer's instructions, or any other suitabletransfection technique known in the art. Suitable transfection markersinclude, for example, β-galactosidase or green fluorescent protein (GFP)expression plasmids or any plasmid that does not contain the samedetectable marker as the antigen-complementary affinity molecule fusionprotein. The fusion protein expressing cells can be sorted and furthercultured, or the tagged antigen-complementary affinity molecule fusionprotein can be purified. In some embodiments, an antigen-complementaryaffinity molecule fusion protein is amplified with a signal peptide. Inalternative embodiments, a cDNA encoding an antigen-complementaryaffinity molecule fusion protein can be amplified without the signalpeptide and subcloned into a vector (pSecTagHis) having a strongsecretion signal peptide. In another example, antigen-complementaryaffinity molecule fusion protein can have an alkaline phosphatase (AP)tag, or a histadine (His) tag for purification. Any method known topersons of ordinary skill in the art for protein purification of theantigen and/or antigen-complementary affinity molecule fusion protein isencompassed for use in the methods of the invention.

In some embodiments, any of the polypeptides described herein isproduced by expression from a recombinant baculovirus vector. In anotherembodiment, any of the polypeptides described herein is expressed by aninsect cell. In yet another embodiment, any of the polypeptidesdescribed herein is isolated from an insect cell. There are severalbenefits of protein expression with baculovirus in insect cells,including high expression levels, ease of scale-up, production ofproteins with post-translational modifications, and simplified cellgrowth. Insect cells do not require CO2 for growth and can be readilyadapted to high-density suspension culture for large-scale expression.Many of the post-translational modification pathways present inmammalian systems are also utilized in insect cells, allowing theproduction of recombinant protein that is antigenically,immunogenically, and functionally similar to the native mammalianprotein.

Baculoviruses are DNA viruses in the family Baculoviridae. These virusesare known to have a narrow host-range that is limited primarily toLepidopteran species of insects (butterflies and moths). The baculovirusAutographa californica Nuclear Polyhedrosis Virus (AcNPV), which hasbecome the prototype baculovirus, replicates efficiently in susceptiblecultured insect cells. AcNPV has a double-stranded closed circular DNAgenome of about 130,000 base-pairs and is well characterized with regardto host range, molecular biology, and genetics. The BaculovirusExpression Vector System (BEVS) is a safe and rapid method for theabundant production of recombinant proteins in insect cells and insects.Baculovirus expression systems are powerful and versatile systems forhigh-level, recombinant protein expression in insect cells. Expressionlevels up to 500 mg/l have been reported using the baculovirusexpression system, making it an ideal system for high-level expression.Recombinant baculoviruses that express foreign genes are constructed byway of homologous recombination between baculovirus DNA and chimericplasmids containing the gene sequence of interest. Recombinant virusescan be detected by virtue of their distinct plaque morphology andplaque-purified to homogeneity.

Recombinant fusion proteins described herein can be produced in insectcells including, but not limited to, cells derived from the Lepidopteranspecies S. frugiperda. Other insect cells that can be infected bybaculovirus, such as those from the species Bombyx mori, Galleriamellanoma, Trichplusia ni, or Lamanthria dispar, can also be used as asuitable substrate to produce recombinant proteins described herein.Baculovirus expression of recombinant proteins is well known in the art.See U.S. Pat. Nos. 4,745,051; 4,879,236; 5,179,007; 5,516,657;5,571,709; 5,759,809. It will be understood by those skilled in the artthat the expression system is not limited to a baculovirus expressionsystem. What is important is that the expression system directs theN-glycosylation of expressed recombinant proteins. The recombinantproteins described herein can also be expressed in other expressionsystems such as Entomopox viruses (the poxviruses of insects),cytoplasmic polyhedrosis viruses (CPV), and transformation of insectcells with the recombinant gene or genes constitutive expression. A goodnumber of baculovirus transfer vectors and the correspondingappropriately modified host cells are commercially available, forexample, pAcGP67, pAcSECG2TA, pVL1392, pVL1393, pAcGHLT, and pAcAB4 fromBD Biosciences; pBAC-3, pBAC-6, pBACgus-6, and pBACsurf-1 from NOVAGEN®,and pPolh-FLAG and pPolh-MAT from SIGMA ALDRICH®.

The region between the promoter and the transcriptional terminator canhave multiple restriction enzyme digestion sites for facilitatingcloning of the foreign coding sequence, in this instance, the coding DNAsequence for an antigen polypeptide, and a complementary affinitymolecule. Additional sequences can be included, e.g., signal peptidesand/or tag coding sequences, such as His-tag, MAT-Tag, FLAG tag,recognition sequence for enterokinase, honeybee melittin secretionsignal, beta-galactosidase, glutathione S-transferase (GST) tag upstreamof the MCS for facilitating the secretion, identification, properinsertion, positive selection of recombinant virus, and/or purificationof the recombinant protein.

In some embodiments, the fusion protein can comprise an N-terminalsignal sequence as disclosed herein. In some embodiments, the signalsequence is attached to the N-terminal of the complementary affinitymolecule as disclosed herein.

In some embodiments, a fusion polypeptide as described herein has aspacer peptide, e.g., a 14-residue spacer (GSPGISGGGGGILE) (SEQ ID NO:41) separating antigen from the complementary affinity molecule. Thecoding sequence of such a short spacer can be constructed by annealing acomplementary pair of primers. One of skill in the art can design andsynthesize oligonucleotides that will code for the selected spacer.Spacer peptides should generally have non-polar amino acid residues,such as glycine and proline.

Standard techniques known to those of skill in the art can be used tointroduce mutations (to create amino acid substitutions in an antigenpolypeptide sequence of the fusion polypeptide described herein, e. g.,in the antigen in the nucleotide sequence encoding the fusionpolypeptide described herein, including, for example, site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, the variant fusionpolypeptide has less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions, inclusive, relative to the fusion polypeptidesdescribed herein.

Certain silent or neutral missense mutations can also be made in the DNAcoding sequence that do not change the encoded amino acid sequence orthe capability to promote transmembrane delivery. These types ofmutations are useful to optimize codon usage, or to improve recombinantprotein expression and production.

Specific site-directed mutagenesis of a coding sequence for the fusionpolypeptide in a vector can be used to create specific amino acidmutations and substitutions. Site-directed mutagenesis can be carriedout using, e. g., the QUICKCHANGE® site-directed mutagenesis kit fromStratagene according to the manufacturer's instructions.

In one embodiment, described herein are expression vectors comprisingthe coding DNA sequence for the polypeptides described herein for theexpression and purification of the recombinant polypeptide produced froma protein expression system using host cells selected from, e.g.,bacteria, mammalian, insect, yeast, or plant cells. The expressionvector should have the necessary 5′ upstream and 3′ downstreamregulatory elements such as promoter sequences, ribosome recognition andTATA box, and 3′ UTR AAUAAA transcription termination sequence forefficient gene transcription and translation in its respective hostcell. The expression vector is, preferably, a vector having thetranscription promoter selected from a group consisting of CMV(cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, β-actinpromoter, SV40 (simian virus 40) promoter and muscle creatine kinasepromoter, and the transcription terminator selected from a groupconsisting of SV40 poly(A) and BGH terminator; more preferably, anexpression vector having the early promoter/enhancer sequence ofcytomegalovirus and the adenovirus tripartite leader/intron sequence andcontaining the replication orgin and poly(A) sequence of SV40. Theexpression vector can have additional coding regions, such as thoseencoding, for example, 6×-histidine, V5, thioredoxin,glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose bindingpeptide, metal-binding peptide, HA and “secretion” signals (Honeybeemelittin, □ α-factor, PHO, Bip), which can be incorporated into theexpressed fusion polypeptide. In addition, there can be enzyme digestionsites incorporated after these coding regions to facilitate theirenzymatic removal if they are not needed. These additional nucleic acidsare useful for the detection of fusion polypeptide expression, forprotein purification by affinity chromatography, enhanced solubility ofthe recombinant protein in the host cytoplasm, and/or for secreting theexpressed fusion polypeptide out into the culture media or thespheroplast of the yeast cells. The expression of the fusion polypeptidecan be constitutive in the host cells or it can be induced, e.g., withcopper sulfate, sugars such as galactose, methanol, methylamine,thiamine, tetracycline, infection with baculovirus, and(isopropyl-beta-D-thiogalactopyranoside) IPTG, a stable synthetic analogof lactose.

In another embodiment, the expression vector comprising a polynucleotidedescribed herein is a viral vector, such as adenovirus, adeno-associatedvirus (AAV), retrovirus, and lentivirus vectors, among others.Recombinant viruses provide a versatile system for gene expressionstudies and therapeutic applications.

In some embodiments, the fusion polypeptides described herein areexpressed from viral infection of mammalian cells. The viral vectors canbe, for example, adenovirus, adeno-associated virus (AAV), retrovirus,and lentivirus. A simplified system for generating recombinantadenoviruses is presented by He et al., 95 PNAS 2509 (1998). The gene ofinterest is first cloned into a shuttle vector, e.g., pAdTrack-CMV. Theresultant plasmid is linearized by digesting with restrictionendonuclease Pmel, and subsequently cotransformed into E. coli. BJ5183cells with an adenoviral backbone plasmid, e.g. pADEASY-1 ofStratagene's ADEASY™ Adenoviral Vector System. Recombinant adenovirusvectors are selected for kanamycin resistance, and recombinationconfirmed by restriction endonuclease analyses. Finally, the linearizedrecombinant plasmid is transfected into adenovirus packaging cell lines,for example HEK 293 cells (E1-transformed human embryonic kidney cells)or 911 (E1-transformed human embryonic retinal cells). Fallaux, et al. 7Human Gene Ther. 215 (1996). Recombinant adenovirus are generated withinthe HEK 293 cells.

Recombinant lentivirus has the advantage of delivery and expression offusion polypeptides in dividing and non-dividing mammalian cells. TheHIV-1 based lentivirus can effectively transduce a broader host rangethan the Moloney Leukemia Virus (MoMLV)-based retroviral systems.Preparation of the recombinant lentivirus can be achieved using, forexample, the pLenti4/V5-DEST™, pLenti6/V5-DEST™ or pLenti vectorstogether with VIRAPOWER™ Lentiviral Expression systems from Invitrogen,Inc.

Recombinant adeno-associated virus (rAAV) vectors are applicable to awide range of host cells including many different human and non-humancell lines or tissues. rAAVs are capable of transducing a broad range ofcell types and transduction is not dependent on active host celldivision. High titers, >10⁸ viral particle/ml, are easily obtained inthe supernatant and 10¹¹-10¹² viral particle/ml with furtherconcentration. The transgene is integrated into the host genome soexpression is long term and stable.

Large scale preparation of AAV vectors is made by a three-plasmidcotransfection of a packaging cell line: AAV vector carrying the codingnucleic acid, AAV RC vector containing AAV rep and cap genes, andadenovirus helper plasmid pDF6, into 50×150 mm plates of subconfluent293 cells. Cells are harvested three days after transfection, andviruses are released by three freeze-thaw cycles or by sonication.

AAV vectors can be purified by two different methods depending on theserotype of the vector. AAV2 vector is purified by the single-stepgravity-flow column purification method based on its affinity forheparin. Auricchio et. al., 12 Human Gene Ther. 71 (2001); Summerford &Samulski, 72 J. Virol. 1438 (1998); Summerford & Samulski, 5 Nat. Med.587 (1999). AAV2/1 and AAV2/5 vectors are currently purified by threesequential CsCl gradients.

Without wishing to be bound to theory, when proteins are expressed by acell, including a bacterial cell, the proteins are targeted to aparticular part in the cell or secreted from the cell. Thus, proteintargeting or protein sorting is the mechanism by which a cell transportsproteins to the appropriate positions in the cell or outside of it.Sorting targets can be the inner space of an organelle, any of severalinterior membranes, the cell's outer membrane, or its exterior viasecretion. This delivery process is carried out based on informationcontained in the protein itself. Correct sorting is crucial for thecell; errors can lead to diseases.

With some exceptions, bacteria lack membrane-bound organelles as foundin eukaryotes, but they may assemble proteins onto various types ofinclusions such as gas vesicles and storage granules. Also, depending onthe species of bacteria, bacteria may have a single plasma membrane(Gram-positive bacteria), or both an inner (plasma) membrane and anouter cell wall membrane, with an aqueous space between the two calledthe periplasm (Gram-negative bacteria). Proteins can be secreted intothe environment, according to whether or not there is an outer membrane.The basic mechanism at the plasma membrane is similar to the eukaryoticone. In addition, bacteria may target proteins into or across the outermembrane. Systems for secreting proteins across the bacterial outermembrane may be quite complex and play key roles in pathogenesis. Thesesystems may be described as type I secretion, type II secretion, etc.

In most Gram-positive bacteria, certain proteins are targeted for exportacross the plasma membrane and subsequent covalent attachment to thebacterial cell wall. A specialized enzyme, sortase, cleaves the targetprotein at a characteristic recognition site near the proteinC-terminus, such as an LPXTG motif (SEQ ID NO: 42) (where X can be anyamino acid), then transfers the protein onto the cell wall. A systemanalogous to sortase/LPXTG, having the motif PEP-CTERM (SEQ ID NO: 43),termed exosortase/PEP-CTERM, is proposed to exist in a broad range ofGram-negative bacteria.

Proteins with appropriate N-terminal targeting signals are synthesizedin the cytoplasm and then directed to a specific protein transportpathway. During, or shortly after its translocation across thecytoplasmic membrane, the protein is processed and folded into itsactive form. Then the translocated protein is either retained at theperiplasmic side of the cell or released into the environment. Since thesignal peptides that target proteins to the membrane are keydeterminants for transport pathway specificity, these signal peptidesare classified according to the transport pathway to which they directproteins. Signal peptide classification is based on the type of signalpeptidase (SPase) that is responsible for the removal of the signalpeptide. The majority of exported proteins are exported from thecytoplasm via the general “Secretory (Sec) pathway”. Most well knownvirulence factors (e.g. exotoxins of Staphylococcus aureus, protectiveantigen of Bacillus anthracis, lysteriolysin 0 of Listeriamonocytogenes) that are secreted by Gram-positive pathogens have atypical N-terminal signal peptide that would lead them to theSec-pathway. Proteins that are secreted via this pathway aretranslocated across the cytoplasmic membrane in an unfolded state.Subsequent processing and folding of these proteins takes place in thecell wall environment on the trans-side of the membrane. In addition tothe Sec system, some Gram-positive bacteria also contain the Tat-systemthat is able to translocate folded proteins across the membrane.Pathogenic bacteria may contain certain special purpose export systemsthat are specifically involved in the transport of only a few proteins.For example, several gene clusters have been identified in mycobacteriathat encode proteins that are secreted into the environment via specificpathways (ESAT-6) and are important for mycobacterial pathogenesis.Specific ATP-binding cassette (ABC) transporters direct the export andprocessing of small antibacterial peptides called bacteriocins. Genesfor endolysins that are responsible for the onset of bacterial lysis areoften located near genes that encode for holin-like proteins, suggestingthat these holins are responsible for endolysin export to the cell wall.Wooldridge, BACT. SECRETED PROTS: SECRETORY MECHS. & ROLE IN PATHOGEN.(Caister Academic Press, 2009)

In some embodiments, the signal sequence useful in the present inventionis OmpA Signal sequence, however any signal sequence commonly known bypersons of ordinary skill in the art which allows the transport andsecretion of antimicrobial agents outside the bacteriophage infectedcell are encompassed for use in the present invention.

Signal sequence that direct secretion of proteins from bacterial cellsare well known in the art, for example as disclosed in Internationalapplication WO 2005/071088. For example, one can use some of thenon-limited examples of signal peptide shown in Table 4, which can beattached to the amino-terminus or carboxyl terminus of the antimicrobialpeptide (Amp) or antimicrobial polypeptide to be expressed by theantimicrobial-agent engineered bacteriophage, e.g., AMP-engineeredbacteriophage. Attachment can be via fusion or chimera composition withselected antigen or antigen-complementary affinity molecule fusionprotein resulting in the secretion from the bacterium infected with theantimicrobial-agent engineered bacteriophage, e.g. AMP-engineeredbacteriophage.

TABLE 4Example signal peptides to direct secretion of a protein or peptide antigen orantigen-complementary affinity molecule fusion protein of a bacterial cellSecretion Signal Peptide Amino Pathway Acid sequence (NH₂-CO₂₎ GeneGenus/Species secA1 MKKIMLVITLILVSPIAQQTEAK Hly (LLO)Listeria monocytogenes D (SEQ ID NO: 44) MKKKIISAILMSTVILSAAAPLSG Usp45Lactococcus lactis VYADT (SEQ ID NO: 45) MKKRKVLIPLMALSTILVSSTGNPag (protective Bacillus anthracis LEVIQAEV (SEQ ID NO: 46) antigen)secA2 MNMKKATIAATAGIAVTAFAAP Iap (invasion- Listeria monocytogenesTIASAST (SEQ ID NO: 47) associated protein p60) MQKTRKERILEALQEEKKNKKSNamA Imo2691 Listeria monocytogenes KKFKTGATIAGVTAIATSITVPGI (autolysin)EVIVSADE (SEQ ID NO: 48) MKKLKMASCALVAGLMFSGLT *BA_0281Bacillus anthracis PNAFAED (SEQ ID NO: 49) (NLP/P60 family)MAKKFNYKLPSMVALTLVGSA *atl (autolysin) Staphylococcus aureusVTAHQVQAAE (SEQ ID NO: 50) Tat MTDKKSENQTEKTETKENKGM Imo0367Listeria monocytogenes TRREMLKLSAVAGTGIAVGATG LGTILNVVDQVDKALT (SEQ IDNO: 51) MAYDSRFDEWVQKLKEESFQN PhoD (alkaline Bacillus subtillisNTFDRRKFIQGAGKIAGLGLGLT phosphatase) IAQSVGAFG (SEQ ID NO: 52)

The polypeptides as described herein, e.g., antigens orantigen-complementary affinity molecule fusion protein can be expressedand purified by a variety methods known to one skilled in the art, forexample, the fusion polypeptides described herein can be purified fromany suitable expression system. Fusion polypeptides can be purified tosubstantial purity by standard techniques, including selectiveprecipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others; which arewell-known in the art. See, e.g., Scopes, PROTEIN PURIFICATION:PRINCIPLES & PRACTICE (1982); U.S. Pat. No. 4,673,641.

A number of procedures can be employed when recombinant proteins arepurified. For example, proteins having established molecular adhesionproperties can be reversibly fused to the protein of choice. With theappropriate ligand, the protein can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,the protein of choice can be purified using affinity or immunoaffinitycolumns.

After the protein is expressed in the host cells, the host cells can belysed to liberate the expressed protein for purification. Methods oflysing the various host cells are featured in “Sample Preparation-Toolsfor Protein Research” EMD Bioscience and in the Current Protocols inProtein Sciences (CPPS). An example purification method is affinitychromatography such as metal-ion affinity chromatograph using nickel,cobalt, or zinc affinity resins for histidine-tagged fusionpolypeptides. Methods of purifying histidine-tagged recombinant proteinsare described by Clontech using their TALON® cobalt resin and byNOVAGEN® in their pET system manual, 10th edition. Another preferredpurification strategy is immuno-affinity chromatography, for example,anti-myc antibody conjugated resin can be used to affinity purifymyc-tagged fusion polypeptides. When appropriate protease recognitionsequences are present, fusion polypeptides can be cleaved from thehistidine or myc tag, releasing the fusion polypeptide from the affinityresin while the histidine-tags and myc-tags are left attached to theaffinity resin.

Standard protein separation techniques for purifying recombinant andnaturally occurring proteins are well known in the art, e.g., solubilityfractionation, size exclusion gel filtration, and various columnchromatography.

Solubility Fractionation:

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the protein of interest. The preferred salt is ammonium sulfate.Ammonium sulfate precipitates proteins by effectively reducing theamount of water in the protein mixture. Proteins then precipitate on thebasis of their solubility. The more hydrophobic a protein is, the morelikely it is to precipitate at lower ammonium sulfate concentrations. Atypical protocol includes adding saturated ammonium sulfate to a proteinsolution so that the resultant ammonium sulfate concentration is between20-30%. This concentration will precipitate the most hydrophobic ofproteins. The precipitate is then discarded (unless the protein ofinterest is hydrophobic) and ammonium sulfate is added to thesupernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Exclusion Filtration:

The molecular weight of the protein of choice can be used to isolate itfrom proteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, AMICON® or MILLIPORE®membranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography:

The protein of choice can also be separated from other proteins on thebasis of its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against recombinant or naturallyoccurring proteins can be conjugated to column matrices and the proteinsimmunopurified. All of these methods are well known in the art. It willbe apparent to one of skill that chromatographic techniques can beperformed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech). For example, an antigenpolypeptide can be purified using a PA63 heptamer affinity column. Singhet al., 269, J. Biol. Chem. 29039 (1994).

In some embodiments, a combination of purification steps comprising, forexample: (a) ion exchange chromatography, (b) hydroxyapatitechromatography, (c) hydrophobic interaction chromatography, and (d) sizeexclusion chromatography can be used to purify the fusion polypeptidesdescribed herein.

Cell-free expression systems are also contemplated. Cell-free expressionsystems offer several advantages over traditional cell-based expressionmethods, including the easy modification of reaction conditions to favorprotein folding, decreased sensitivity to product toxicity andsuitability for high-throughput strategies such as rapid expressionscreening or large amount protein production because of reduced reactionvolumes and process time. The cell-free expression system can useplasmid or linear DNA. Moreover, improvements in translation efficiencyhave resulted in yields that exceed a milligram of protein permilliliter of reaction mix. Commercially available cell-free expressionsystems include the TNT coupled reticulocyte lysate Systems (Promega)which uses rabbit reticulocyte-based in vitro system.

Formulations of an Immune Composition and Methods of Use

Specific embodiments of the present invention provide for use of theimmunogenic compositions as disclosed herein to elicit an immuneresponse in an animal. More specifically, the compositions elicit bothhumoral and cellular immunity, and in many instance mucosal immunity.Embodiments of the present invention provide at least partial protectionfrom or partial improvement after infection by, in particular,pneumococcus. Pneumococci cause a number of diseases, such asmeningitis, pneumonia, bacteremia, and otitis media. Almost one millionchildren die of pneumococcal diseases worldwide every year. S.pneumoniae have been studied extensively, and at least some of thegenomes sequenced. See, e.g., U.S. Pat. No. 7,141,418. Althoughantibodies to the capsular polysaccharides, which define the knownserotypes, confer serotype-specific protection, other protectivemechanisms of immunity have been described. See Malley et al., 88 J.Mol. Med. 135 (2010). These other protective mechanisms include, but arenot limited to, antibodies to noncapsular antigens and T cell responsesto pneumococcal constituents. The application of protein-polysaccharideconjugate vaccine, PCV7, has reduced diseases significantly. Black etal., 24(S2) Vaccine 79 (2006); Hansen et al., 25 Pediatr. Infect. Dis.J. 779 (2006)). Yet, recent studies have shown that the lack of otherserotypes in PCV7 has resulted in emerging replacement pneumococcalserotypes. Pichichero & Casey, 26(S10) Pediatr. Infect. Dis. J. S12(2007).

Certain pneumococcal antigens common to all serotypes of the specieshave been shown to have immunoprotective potential despite theencapsulation, e.g., the surface proteins PspA, PspC, PsaA and thecytotoxin pneumolysin or pneumolysoid mutants (Basset et al., 75 Infect.Immun. 5460 (2007); Briles et al., 18 Vaccine 1707 (2000)); the use ofgenomics and mutational libraries has identified several dozenadditional species-common proteins (Hava & Camilli, 45 Mol. Microbiol.1389 (2002); Wizemann et al., 60 Infect. Immun. 1593 (2001)). Immunityhas been induced by individual antigens in animal models (Alexander etal., 62 Infect. Immun. 5683 (1994); Balachandran et al., 70 Infect.Immun. 2526 (2002); Chung et al., 170 J. Immunol. 1958 (2003); Glover etal., 76 Infect. Immun. 2767 (2008); Wu et al., 175 J. Infect. Dis. 839(1997)), but no vaccine based on a common antigen has been approved forhuman use to date.

In one embodiment, provided herein is a method of vaccinating a mammalcomprising administering the immunogenic composition comprising at leastone, or multiple antigens attached to at least one type of polymerscaffold, e.g., a polysaccharide or carbohydrate polymer for use ineliciting an immune response to the one or more antigens attached to thepolymer when administered to a subject. In some embodiments, the immuneresponse is a humoral and/or cellular immune response.

Accordingly, one aspect of the present invention relates to methods toelicit an immune response in a subject, comprising administering to thesubject an immunogenic composition comprising at least one type of thepolymer, e.g., a polysaccharide, at least one antigen, and at least onecomplementary affinity-molecule pair comprising (i) a first affinitymolecule which associates with the polymer, e.g., a polysaccharide, and(ii) a complementary affinity molecule which associates with theantigen, to attach the antigen to the polymer, e.g., a polysaccharide,(e.g., the first affinity molecule associates with the complementaryaffinity molecule to link the antigen to the polymer, e.g.,polysaccharide).

Accordingly, one aspect of the present invention relates to methods toelicit a humoral and/or cellular immunity to multiple antigens at thesame time, e.g., where the immunogenic composition administered to thesubject comprises a polymer comprising at least 1, or at least 2, or amore, e.g., a plurality of the same or different antigens.

One aspect of the present invention relates to a method of immunizationor vaccinating a subject, e.g., a bird or a mammal, e.g., a humanagainst a pathogen comprises administering an immune composition asdisclosed herein comprising at least one antigen derived from one ormore pathogens. In some embodiments, a subject can be immunized againstat least 1, or at least 2, or at least 2, or at least 3, or at least 5,or at least 10, or at least 15, or at least about 20, or at least 50, orat least about 100, or more than 100 different pathogens at the sametime, where the polymer of the immunogenic composition as thecorresponding different antigens attached.

In some embodiments, a subject can be administered several differentimmunogenic compositions as disclosed herein, for example, a subject canbe administered a composition comprising a polymer with an antigen, or aplurality of antigens, e.g., antigens A, B, C, and D etc., and alsoadministered a composition comprising a polymer comprising a differentantigen, or a different set of antigens, e.g., antigens W, X, Y, and Zetc. Alternatively, a subject can be administered a compositioncomprising a polymer A with an antigen, or a plurality of antigens,e.g., antigens A, B, C, and D, etc., and also administered a compositioncomprising a polymer B comprising the same e.g., antigens A, B, C, and Detc., or a different set of antigens. It is envisioned that the presentinvention provides a methods for the immunization of a subject with asmany antigens as desired, e.g., with a variety of different immunogeniccomplexes as described herein, to enable immunization with as many as100 or more antigens.

In one embodiment, the immunogenic compositions as described hereincomprise a pharmaceutically acceptable carrier. In another embodiment,the immunogenic composition composition described herein is formulatedfor administering to a bird, mammal, or human, as or in a vaccine.Suitable formulations can be found in, for example, Remington'sPharmaceutical Sciences (2006), or Introduction to Pharmaceutical DosageForms (4th ed., Lea & Febiger, Philadelphia, 1985).

In one embodiment, the immunogenic compositions as described hereincomprise pharmaceutically acceptable carriers that are inherentlynontoxic and nontherapeutic. Examples of such carriers include ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, and polyethyleneglycol. For all administrations, conventional depot forms are suitablyused. Such forms include, for example, microcapsules, nano-capsules,liposomes, plasters, inhalation forms, nose sprays, sublingual tablets,and sustained release preparations. For examples of sustained releasecompositions, see U.S. Pat. Nos. 3,773,919, 3,887,699, EP 58,481A, EP158,277A, Canadian Patent No. 1176565; Sidman et al., 22 Biopolymers 547(1983); Langer et al., 12 Chem. Tech. 98 (1982). The proteins willusually be formulated at a concentration of about 0.1 mg/ml to 100 mg/mlper application per patient.

In one embodiment, other ingredients can be added to vaccineformulations, including antioxidants, e.g., ascorbic acid; low molecularweight (less than about ten residues) polypeptides, e.g., polyarginineor tripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids, such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose, or dextrins; chelatingagents such as EDTA; and sugar alcohols such as mannitol or sorbitol.

In some embodiments, the present MAPS immunogen compositions areadministered with at least one adjuvant. Adjuvants are a heterogeneousgroup of substances that enhance the immunological response against anantigen that is administered simultaneously. In some instances,adjuvants improve the immune response so that less vaccine is needed.Adjuvants serve to bring the antigen—the substance that stimulates thespecific protective immune response—into contact with the immune systemand influence the type of immunity produced, as well as the quality ofthe immune response (magnitude or duration). Adjuvants can also decreasethe toxicity of certain antigens; and provide solubility to some vaccinecomponents. Almost all adjuvants used today for enhancement of theimmune response against antigens are particles or form particlestogether with the antigen. In the book VACCINE DESIGN—SUBUNIT & ADJUVANTAPPROACH (Powell & Newman, Eds., Plenum Press, 1995), many knownadjuvants are described both regarding their immunological activity andregarding their chemical characteristics. The type of adjuvants that donot form particles are a group of substances that act as immunologicalsignal substances and that under normal conditions consist of thesubstances that are formed by the immune system as a consequence of theimmunological activation after administration of particulate adjuvantsystems.

Adjuvants for immunogenic compositions and vaccines are well known inthe art. Examples include, but not limited to, monoglycerides and fattyacids (e. g. a mixture of mono-olein, oleic acid, and soybean oil);mineral salts, e.g., aluminium hydroxide and aluminium or calciumphosphate gels; oil emulsions and surfactant based formulations, e.g.,MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS21(purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21),MPL-SE, Montanide ISA-51 and ISA-720 (stabilised water-in-oil emulsion);particulate adjuvants, e.g., virosomes (unilamellar liposomal vehiclesincorporating influenza haemagglutinin), ASO4 ([SBAS4] Al salt withMPL), ISCOMS (structured complex of saponins and lipids), polylactideco-glycolide (PLG); microbial derivatives (natural and synthetic), e.g.,monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton),AGP [RC-529] (synthetic acylated monosaccharide), Detox-PC, DC_Chol(lipoidal immunostimulators able to self-organize into liposomes),OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotidescontaining immunostimulatory CpG motifs), or other DNA structures,modified LT and CT (genetically modified bacterial toxins to providenon-toxic adjuvant effects); endogenous human immunomodulators, e.g.,hGM-CSF or hIL-12 (cytokines that can be administered either as proteinor plasmid encoded), Immudaptin (C3d tandem array), MoGM-CSF,TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I,GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59and inert vehicles, such as gold particles. Additional adjuvants areknown in the art, see, e.g., U.S. Pat. No. 6,890,540; U. S. Patent Pub.No. 2005;0244420; PCT/SE97/01003.

In some embodiments an adjuvant is a particulate and can have acharacteristic of being slowly biodegradable. Care must be taken toensure that that the adjuvant do not form toxic metabolites. Preferably,in some embodiments, such adjuvants can be matrices used are mainlysubstances originating from a body. These include lactic acid polymers,poly-amino acids (proteins), carbohydrates, lipids and biocompatiblepolymers with low toxicity. Combinations of these groups of substancesoriginating from a body or combinations of substances originating from abody and biocompatible polymers can also be used. Lipids are thepreferred substances since they display structures that make thembiodegradable as well as the fact that they are a critical element inall biological membranes.

In one embodiment, the immunogenic compositions as described herein foradministration must be sterile for administration to a subject.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes), or by gammairradiation.

In some embodiments, the immunogenic compositions described hereinfurther comprise pharmaceutical excipients including, but not limited tobiocompatible oils, physiological saline solutions, preservatives,carbohydrate, protein, amino acids, osmotic pressure controlling agents,carrier gases, pH-controlling agents, organic solvents, hydrophobicagents, enzyme inhibitors, water absorbing polymers, surfactants,absorption promoters and anti-oxidative agents. Representative examplesof carbohydrates include soluble sugars such as hydropropyl cellulose,carboxymethyl cellulose, sodium carboxyl cellulose, hyaluronic acid,chitosan, alginate, glucose, xylose, galactose, fructose, maltose,saccharose, dextran, chondroitin sulfate, etc. Representative examplesof proteins include albumin, gelatin, etc. Representative examples ofamino acids include glycine, alanine, glutamic acid, arginine, lysine,and their salts. Such pharmaceutical excipients are well-known in theart.

In some embodiments, the immunogenic MAPS composition is administered incombination with other therapeutic ingredients including, e.g.,γ-interferon, cytokines, chemotherapeutic agents, or anti-inflammatory,or anti-viral agents. In some embodiments, the immunogenic compositionas disclosed herein can be administered with one or more co-stimulatorymolecules and/or adjuvants as disclosed herein.

In some embodiments, the immunogenic composition is administered in apure or substantially pure form, but may be administered as apharmaceutical composition, formulation or preparation. Such formulationcomprises MAPS described herein together with one or morepharmaceutically acceptable carriers and optionally other therapeuticingredients. Other therapeutic ingredients include compounds thatenhance antigen presentation, e.g., gamma interferon, cytokines,chemotherapeutic agents, or anti-inflammatory agents. The formulationscan conveniently be presented in unit dosage form and may be prepared bymethods well known in the pharmaceutical art. For example, Plotkin andMortimer, in VACCINES (2nd ed., W.B. Saunders Co., 1994) describesvaccination of animals or humans to induce an immune response specificfor particular pathogens, as well as methods of preparing antigen,determining a suitable dose of antigen, and assaying for induction of animmune response.

Formulations suitable for intravenous, intramuscular, intranasal, oral,sublingual, vaginal, rectal, subcutaneous, or intraperitonealadministration conveniently comprise sterile aqueous solutions of theactive ingredient with solutions which are preferably isotonic with theblood of the recipient. Such formulations may be conveniently preparedby dissolving solid active ingredient in water containingphysiologically compatible substances such as sodium chloride (e.g.,0.1M-2.0 M), glycine, and the like, and having a buffered pH compatiblewith physiological conditions to produce an aqueous solution, andrendering the solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Formulations for an intranasal delivery are described in U.S. Pat. Nos.5,427,782; 5,843,451; 6,398,774.

The formulations of the immunogenic compositions can incorporate astabilizer. Illustrative stabilizers are polyethylene glycol, proteins,saccharide, amino acids, inorganic acids, and organic acids which may beused either on their own or as admixtures. Two or more stabilizers maybe used in aqueous solutions at the appropriate concentration and/or pH.The specific osmotic pressure in such aqueous solution is generally inthe range of 0.1-3.0 osmoses, preferably in the range of 0.80-1.2. ThepH of the aqueous solution is adjusted to be within the range of pH5.0-9.0, preferably within the range of pH 6-8.

When oral preparations are desired, the immunogenic compositions can becombined with typical carriers, such as lactose, sucrose, starch, talcmagnesium stearate, crystalline cellulose, methyl cellulose,carboxymethyl cellulose, glycerin, sodium alginate or gum arabic amongothers.

In some embodiments, the immunogenic compositions as described hereincan be administered intravenously, intranasally, intramuscularly,subcutaneously, intraperitoneally, sublingually, vaginal, rectal ororally. In some embodiments, the route of administration is oral,intranasal, subcutaneous, or intramuscular. In some embodiments, theroute of administration is intranasal administration.

Vaccination can be conducted by conventional methods. For example, animmunogenic compositions can be used in a suitable diluent such assaline or water, or complete or incomplete adjuvants. The immunogeniccomposition can be administered by any route appropriate for elicitingan immune response. The immunogenic composition can be administered onceor at periodic intervals until an immune response is elicited. Immuneresponses can be detected by a variety of methods known to those skilledin the art, including but not limited to, antibody production,cytotoxicity assay, proliferation assay and cytokine release assays. Forexample, samples of blood can be drawn from the immunized mammal, andanalyzed for the presence of antibodies against the antigens of theimmunogenic composition by ELISA (see de Boer et. al., 115 Arch Virol.147 (1990) and the titer of these antibodies can be determined bymethods known in the art.

The precise dose to be employed in the formulation will also depend onthe route of administration and should be decided according to thejudgment of the practitioner and each patient's circumstances. Forexample, a range of 25 μg-900 μg total protein can be administeredmonthly for three months.

Ultimately, the attending physician will decide the amount ofimmunogenic composition or vaccine composition to administer toparticular individuals. As with all immunogenic compositions orvaccines, the immunologically effective amounts of the immunogens mustbe determined empirically. Factors to be considered include theimmunogenicity, whether or not the immunogen will be complexed with orcovalently attached to an adjuvant or carrier protein or other carrier,routes of administrations and the number of immunizing dosages to beadministered. Such factors are known in the vaccine art and it is wellwithin the skill of immunologists to make such determinations withoutundue experimentation.

In one embodiment, an immunogenic composition or vaccine composition asdescribed herein, when administered to mice, can provoke an immuneresponse that prevents a disease symptom in at least 20% of animalschallenged with 5 LD₅₀ of the immunogenic composition comprisingantigens to which the disease symptom is prevented. Methods ofvaccination and challenging an immunized animal are known to one skilledin the art. For example, a 10 μg aliquot of an immunogenic compositionor vaccine composition as disclosed herein can be prepared in 100 μl PBSand/or with addition of incomplete Freund's adjuvant and injectedintramuscularly per mouse per vaccination. Alternatively, parenteral,intraperitoneal and footpad injections can be used. Volumes of footpadinjections are reduced to 50 Mice can be immunized with an immunogeniccomposition or vaccine composition as disclosed herein on three separateoccasions with several days, e.g., 14 days interval in between.

Efficacy of vaccination can be tested by challenge with the pathogen.Seven days after the last dose of an immunogenic composition, theimmunized mice are challenged intranasally with a pathogenic organismfrom which the antigen was derived. Ether anaesthetized mice (10 g to 12g) can be infected intranasally with 50 μl of PBS-diluted allantoicfluid containing 5 LD₅₀ of the pathogenic organism. Protection can bemeasured by monitoring animal survival and body weight, which isassessed throughout an observation period of 21 days. Severely affectedmice are euthanized One LD₅₀ of A/Mallard/Pennsylvania/10218/84 is equalto 100-1000 the Tissue Culture Infectious Dose50 (TCID50) assay.

In other embodiments, the immunized mice can be challenged with avariety of different pathogenic organisms, e.g., different pathogenicorganisms from which each of the antigens attached to the polymer arederived. For example, of an immunogenic composition comprises fivedifferent antigens attached to the polymer, e.g., polysaccharide, whereeach antigen is derived from five different pathogenic organisms, theimmunized mice can be challenged with each of the five differentpathogenic organisms, either sequentially (in any order) orconcurrently. One skilled in the art would be able to determine the LD50for each pathogenic organism used to challenge the immunized mice bymethods known in the art. See, e.g., LaBarre & Lowy, 96 J. Virol. Meths.107 (2001); Golub, 59J. Immunol. 7 (1948).

Kits

The present invention also provides for kits for producing animmunogenic composition as disclosed herein which is useful for aninvestigator to tailor an immunogenic composition with their preferredantigens, e.g., for research purposes to assess the effect of anantigen, or a combination of antigens on immune response. Such kits canbe prepared from readily available materials and reagents. For example,such kits can comprise any one or more of the following materials: acontainer comprising a polymer, e.g., a polysaccharide, cross-linkedwith a plurality of first affinity molecules; and a container comprisinga complementary affinity molecule which associates with the firstaffinity molecule, wherein the complementary affinity moleculeassociates with an antigen.

In another embodiment, the kit can comprise a container comprising apolymer, e.g., a polysaccharide, a container comprising a plurality offirst affinity molecules, and a container comprising a cross-linkingreagent for cross-linking the first affinity molecules to the polymer.

In some embodiments, the kit further comprises a means to attach thecomplementary affinity molecule to the antigen, where the means can beby a cross-linking reagent or by some intermediary fusion protein. Insome embodiments, the kit can comprise at least one co-stimulationfactor which can be added to the polymer. In some embodiments, the kitcomprises a cross-linking reagent, for example, but not limited to, CDAP(1-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sodiumcyanoborohydride; cyanogen bromide; ammonium bicarbonate/iodoacetic acidfor linking the co-factor to the polymer.

A variety of kits and components can be prepared for use in the methodsdescribed herein, depending upon the intended use of the kit, theparticular target antigen and the needs of the user.

The invention can be further described any of the embodiments in thefollowing numbered paragraphs:

-   1. A soluble biotin-binding protein, comprising an amino acid    sequence

(SEQ ID NO: 1) FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDand any functional derivatives thereof.

-   2. The biotin-binding of paragraph 1, wherein the biotin-binding    protein is produced in soluble form at a level of at least 10 mg/L    of culture media in E. coli.-   3. The biotin binding protein of any of paragraphs 1-2, wherein the    biotin-binding protein is a dimer.-   4. The biotin-binding protein of any of paragraphs 1-3, wherein the    biotin-binding protein comprises a bacterial signal sequence at the    N-terminus.-   5. The biotin-binding protein of paragraph 4, wherein the bacterial    signal sequence is

(SEQ ID No: 2) MKKIWLALAGLVLAFSASA.

-   6. The biotin-binding protein of any of paragraph 4 or 5, wherein    the signal sequence is linked to the biotin-protein by a peptide    linker.-   7. The biotin-binding protein of paragraph 6, wherein the peptide    linker comprises the amino acid sequence AQDP (SEQ ID NO: 8) or VSDP    (SEQ ID NO: 9).-   8. The biotin-binding protein of any of paragraphs 1-7, wherein the    biotin-binding protein comprises a purification tag at the    C-terminus.-   9. The biotin-binding protein of paragraph 8, wherein the    purification tag is selected from the group consisting of a    histidine tag, a c-my tag, a Halo tag, a Flag tag, and any    combinations thereof.-   10. The biotin-binding protein of paragraph 9, wherein the histidine    tag comprises the amino acid sequence HHHHHH (SEQ ID NO: 10).-   11. The biotin-binding protein of any of paragraphs 8-10, wherein    the purification tag is linked to the biotin-binding protein via a    peptide linker.-   12. The biotin-binding protein of paragraph 11, where the peptide    linker comprises the amino acid sequence VDKLAAALE (SEQ ID NO: 11)    or GGGGSSSVDKLAAALE (SEQ ID NO: 12).-   13. The biotin-binding protein of any of paragraph 1-12, wherein the    biotin-binding protein comprises the amino acid sequence

(SEQ ID NO: 15) MKKIWLALAGLVLAFSASAAQDPFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSVDKLAAALEHHHHHH.

-   14. A composition comprising a biotin-binding protein of any of    paragraphs 1-13.-   15. A fusion protein comprising a biotin-binding protein and a    protein or a peptide.-   16. The fusion protein of paragraph 15, wherein the protein or    peptide is fused to the biotin-binding protein by a peptide linker.-   17. The fusion protein of paragraph 15, wherein the peptide linker    comprises the amino acid sequence GGGGSSS (SEQ ID NO: 22).-   18. The fusion protein of any of paragraphs 15-17, wherein the    protein or peptide is an antigen selected from the group consisting    of: pneumococcal antigens, tuberculosis antigens, anthrax antigens,    HIV antigens, seasonal or epidemic flu antigens, influenzae    antigens, Pertussis antigens, Staphylococcus aureus antigens,    Meningococcal antigens, Haemophilus antigens, HPV antigens, or    combinations thereof.-   19. The fusion protein of any of paragraphs 15-18, wherein the    antigen is a non-hemolytic variant of S. aureus alpha-hemolysin.-   20. The fusion of paragraph 19, wherein the non-hemolytic variant    of S. aureus alpha-hemolysin comprises a mutation at amino acid    residue 205, 213 or 209-211 of wild-type S. aureus alpha-hemolysin.-   21. The fusion protein of paragraph 19, wherein the non-hemolytic    variant of S. aureus alpha-hemolysin comprises one of the following    mutations in the wild-type S. aureus alpha-hemolysin: (i) residue    205 W to A; (ii) residue 213 W to A; or (iii) residues 209-211 DRD    to AAA.-   22. The fusion protein of paragraph 19, wherein the non-hemolytic    variant of S. aureus alpha-hemolysin comprises the amino acid    sequence selected from the group consisting of:

(i) W205A (SEQ ID NO: 23)ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNAGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN; (ii) W213A (SEQ ID NO: 24)ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSANPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN; (iii) DRD209-211AAA(SEQ ID NO: 25) ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYAAASWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSMWKGTNTKDKWIDRSSERYKIDWEKEEMTN;and functional variants, portions, and derivatives thereof

-   23. The fusion protein of any of paragraphs 15-22, wherein the    fusion protein comprises a bacterial signal sequence at the    N-terminus.-   24. The fusion protein of paragraph 23, wherein the bacterial signal    sequence is MKKIWLALAGLVLAFSASA (SEQ ID No: 2).-   25. The fusion protein of any of paragraph 23 or 24, wherein the    signal sequence is linked to the biotin-protein by a peptide linker.-   26. The fusion protein of paragraph 25, wherein the peptide linker    comprises the amino acid sequence AQDP (SEQ ID NO: 8) or VSDP (SEQ    ID NO: 9).-   27. The fusion protein of any of paragraphs 15-26, wherein the    fusion protein comprises a purification tag at the C-terminus.-   28. The fusion protein of paragraph 27, wherein the purification tag    is selected from the group consisting of a histidine tag, a c-my    tag, a Halo tag, a Flag tag, and any combinations thereof.-   29. The fusion protein of paragraph 27, wherein the histidine tag    comprises the amino acid sequence HHHHHH (SEQ ID NO: 10).-   30. The fusion protein of any of paragraphs 27-29, wherein the    purification tag is linked to the biotin-binding protein via a    peptide linker.-   31. The fusion protein of paragraph 30, where the peptide linker    comprises the amino acid sequence VDKLAAALE (SEQ ID NO: 11).-   32. The fusion protein of any of paragraphs 15-31, wherein the    biotin-binding protein is a biotin-binding protein of any of    paragraphs 1-13.-   33. The fusion protein of any of paragraphs 15-32, wherein the    fusion protein comprises the amino acid sequence selected from the    group consisting of:

(i) W205A (SEQ ID NO: 26)MKKIWLALAGLVLAFSASAAQDPFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNAGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTNVDKLAAALEHHHHHH; (ii) W213A (SEQ ID NO: 27)MKKIWLALAGLVLAFSASAAQDPFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSANPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSMWKGTNTKDKWIDRSSERYKIDWEKEEMTNVDKLAAALEHHHHHH; (iii) DRD209-211AA (SEQ ID NO: 28)MKKIWLALAGLVLAFSASAAQDPFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYAAASWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSMWKGTNTKDKWIDRSSERYKIDWEKEEMTNVDKLAAALEHHHHHH.

34. A composition comprising a fusion protein of any of paragraphs15-33.

-   35. A mutant alpha-hemolysin (mHla) protein, comprising a mutation    at amino acid residue 205, 213 or 209-211 of wild-type S. aureus    alpha-hemolysin, wherein the mutant alpha-hemolysin has lower    hemolytic activity than an equivalent titer of wild-type    alpha-hemolysin (Hla).-   36. The mutant alpha-hemolysin of paragraph 35, wherein the    hemolytic activity of mutant alpha-hemolysin is at least 25% lower    than an equivalent titer of wild-type Hla.-   37. The mutant alpha-hemolysin of paragraph 35 or 36, wherein the    mutant alpha-hemolysin comprises one of the following mutations in    the wild-type S. aureus alphs-hemolysin: (i) residue 205 W to    A; (ii) residue 213 W to A; or (iii) residues 209-211 DRD to AAA.-   38. The mutant alpha-hemolysin any of paragraphs 13-15, wherein the    mutant alpha-hemolysin comprises an amino acid sequence selected    from the group consisting of:

(i) W205A (SEQ ID NO: 23)ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNAGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN; (ii) W213A (SEQ ID NO: 24)ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSANPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN; (iii) DRD209-211AAA(SEQ ID NO: 25) ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYAAASWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSMWKGTNTKDKWIDRSSERYKIDWEKEEMTN;and functional variants, portions, and derivatives thereof

-   39. A composition comprising a mutant alpha-hemolysin of any of    paragraphs 35-38.-   40. A fusion protein comprising an alpha-hemolysin and a    biotin-binding domain, wherein the fusion protein has lower    hemolytic activity than an equivalent titer of wild-type    alpha-hemolysin (Hla).-   41. The fusion protein of paragraph 18, wherein the alpha-hemolysin    is a mutant hemolysin of any of paragraphs 35-38 or the    alpha-hemolysin consists of the the amino acids sequence of amino    acids 27-319 of wild-type alpha-hemolysin of S. aureus.-   42. The fusion protein of paragraph 19, wherein the biotin-binding    domain consists of the amino acid sequence SEQ ID NO: 1.-   43. The fusion protein of any of paragraphs 40-42, wherein the    biotin binding domain and the mutant alpha-hemolysin are linked by a    peptide linker.-   44. The fusion protein of paragraph 43, wherein the peptide linker    comprises the amino acid sequence GGGGSSS (SEQ ID NO: 22).-   45. The fusion protein of any of paragraphs 40-44, wherein the    fusion protein comprises a bacterial signal sequence at the    N-terminus.-   46. The fusion protein of paragraph 45, wherein the bacterial signal    sequence is

(SEQ ID No: 2) MKKIWLALAGLVLAFSASA.

-   47. The fusion protein of any of paragraph 45 or 46, wherein the    signal sequence is linked to the biotin-protein by a peptide linker.-   48. The fusion protein of paragraph 47, wherein the peptide linker    comprises the amino acid sequence AQDP (SEQ ID NO: 8) or VSDP (SEQ    ID NO: 9).-   49. The fusion protein of any of paragraphs 40-48, wherein the    fusion protein comprises a purification tag at the C-terminus.-   50. The fusion protein of paragraph 49, wherein the purification tag    is selected from the group consisting of a histidine tag, a c-my    tag, a Halo tag, a Flag tag, and any combinations thereof-   51. The fusion protein of paragraph 50, wherein the histidine tag    comprises the amino acid sequence

(SEQ ID NO: 10) HHHHHH.

52. The fusion protein of any of paragraphs 49-51, wherein thepurification tag is linked to the biotin-binding protein via a peptidelinker.

-   53. The fusion protein of paragraph 52, where the peptide linker    comprises the amino acid sequence

(SEQ ID NO: 11) VDKLAAALE.

54. The fusion protein of any of paragraphs 40-53, wherein thebiotin-binding domain is a biotin-binding protein of any of paragraphs1-13.

-   55. The fusion protein of any of paragraphs 40-54, wherein the    fusion protein comprises the amino acid sequence selected from the    group consisting of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.-   56. The fusion protein of any of paragraphs 40-55, wherein the    hemolytic activity of the fusion protein is at least 25% lower than    an equivalent titer of wild-type Hla.-   57. A composition comprising a fusion protein of any of paragraphs    40-56.-   58. A method inducing an immune response in a subject, comprising    administering to the subject a composition of paragraph 14, 34, 39,    or 57.-   59. A method of vaccinating a mammal against at least one    antigen-bearing pathogen, the method comprising administering a    composition of paragraph 14, 34, 39, or 57.-   60. The method of any of paragraphs 58 or 59, wherein the subject is    a human.-   61. The method of any of paragraphs 58 or 59, wherein the subject is    an agricultural or non-domestic animal.-   62. The method of any of paragraphs 58 or 59, wherein the subject is    a domestic animal.-   63. The method of any of paragraphs 58 or 59, wherein administration    is via subcutaneous, intranasal, intradermal or intra muscular    injection.-   64. The method of paragraph 58, wherein the immune response is an    antibody/B-cell response.-   65. The method of paragraph 58, wherein the immune response is a    CD4+ T-cell response, including Th1, Th2, or Th17 response.-   66. The method of paragraph 58, wherein the immune response is a    CD8+ T-cell response.-   67. The composition of any of paragraphs 14, 34, 39, or 57 for use    in a diagnostic for exposure to a pathogen or immune threat.-   68. A lipidated biotin-binding protein, comprising an amino acid    sequence

(SEQ ID NO: 1) FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDand any functional derivatives thereof.

-   69. The lipidated biotin-binding of paragraph 68, wherein the    biotin-binding protein is produced in soluble form at a level of at    least 10 mg/L of culture media in E. coli.-   70. The lipidated biotin binding protein of any of paragraphs 68-69,    wherein the biotin-binding protein is a dimer.-   71. The lipidated biotin-binding protein of any of paragraphs 68-70,    wherein the biotin-binding protein comprises a lipidation sequence    at the N-terminus.-   72. The lipidated biotin-binding protein of paragraph 71, wherein    the lipidation sequence is

(SEQ ID No: 3) MKKVAAFVALSLLMAGC

-   73. The lipidated biotin-binding protein of any of paragraph 71 or    72, wherein the lipidation sequence is linked to the biotin-protein    by a peptide linker.-   74. The lipidated biotin-binding protein of paragraph 73, wherein    the peptide linker comprises the amino acid sequence AQDP (SEQ ID    NO: 8) or VSDP (SEQ ID NO: 9).-   75. The lipidated biotin-binding protein of any of paragraphs 68-74,    wherein the biotin-binding protein comprises a purification tag at    the C-terminus.-   76. The lipidated biotin-binding protein of paragraph 75, wherein    the purification tag is selected from the group consisting of a    histidine tag, a c-my tag, a Halo tag, a Flag tag, and any    combinations thereof.-   77. The lipidated biotin-binding protein of paragraph 76, wherein    the histidine tag comprises the amino acid sequence HHHHHH (SEQ ID    NO: 10).-   78. The lipidated biotin-binding protein of any of paragraphs 75-77,    wherein the purification tag is linked to the biotin-binding protein    via a peptide linker.-   79. The lipidated biotin-binding protein of paragraph 78, where the    peptide linker comprises the amino acid sequence VDKLAAALE (SEQ ID    NO: 11) or GGGGSSSVDKLAAALE (SEQ ID NO: 12).-   80. The lipidated biotin-binding protein of any of paragraph 68-79,    wherein the biotin-binding protein comprises the amino acid sequence

(SEQ ID NO: 20) MKKVAAFVALSLLMAGCVSDPFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTEN KSLLKD.

-   81. A composition comprising a lipidated biotin-binding protein of    any of paragraphs 68-80.-   82. A fusion protein comprising a lipidated biotin-binding protein    and a protein or a peptide.-   83. The fusion protein of paragraph 82, wherein the protein or    peptide is fused to the lipidated biotin-binding protein by a    peptide linker.-   84. The fusion protein of paragraph 83, wherein the peptide linker    comprises the amino acid sequence GGGGSSS (SEQ ID NO: 22).-   85. The fusion protein of any of paragraphs 82-84, wherein the    protein or peptide is an antigen selected from the group consisting    of: pneumococcal antigens, tuberculosis antigens, anthrax antigens,    HIV antigens, seasonal or epidemic flu antigens, influenzae    antigens, Pertussis antigens, Staphylococcus aureus antigens,    Meningococcal antigens, Haemophilus antigens, HPV antigens, or    combinations thereof.-   86. The fusion protein of any of paragraph 85, wherein the antigen    is a non-hemolytic variant of S. aureus alpha-hemolysin.-   87. The fusion of paragraph 86, wherein the non-hemolytic variant    of S. aureus alpha-hemolysin comprises a mutation at amino acid    residue 205, 213 or 209-211 of wild-type S. aureus alpha-hemolysin.-   88. The fusion protein of paragraph 86, wherein the non-hemolytic    variant of S. aureus alpha-hemolysin comprises one of the following    mutations in the wild-type S. aureus alpha-hemolysin: (i) residue    205 W to A; (ii) residue 213 W to A; or (iii) residues 209-211 DRD    to AAA.-   89. The fusion protein of paragraph 86, wherein the non-hemolytic    variant of S. aureus alpha-hemolysin comprises the amino acid    sequence selected from the group consisting of:

(iv) W205A (SEQ ID NO: 23)ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNAGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN; (v) W213A (SEQ ID NO: 24)ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSANPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN; (vi) DRD209-211AAA(SEQ ID NO: 25) ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYAAASWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN;and functional variants, portions, and derivatives thereof

-   90. The fusion protein of any of paragraphs 82-89, wherein the    fusion protein comprises a lipidation sequence at the N-terminus.-   91. The fusion protein of paragraph 90, wherein the lipidation    sequence is

(SEQ ID No: 3) MKKVAAFVALSLLMAGC.

-   92. The fusion protein of any of paragraph 90 or 91, wherein the    signal sequence is linked to the biotin-protein by a peptide linker.-   93. The fusion protein of paragraph 92, wherein the peptide linker    comprises the amino acid sequence AQDP (SEQ ID NO: 8) or VSDP (SEQ    ID NO: 9).-   94. The fusion protein of any of paragraphs 82-93, wherein the    fusion protein comprises a purification tag at the C-terminus.-   95. The fusion protein of paragraph 94, wherein the purification tag    is selected from the group consisting of a histidine tag, a c-my    tag, a Halo tag, a Flag tag, and any combinations thereof-   96. The fusion protein of paragraph 95, wherein the histidine tag    comprises the amino acid sequence

(SEQ ID NO: 10) HHHHHH.

-   97. The fusion protein of any of paragraphs 93-96, wherein the    purification tag is linked to the biotin-binding protein via a    peptide linker.-   98. The fusion protein of paragraph 97, where the peptide linker    comprises the amino acid sequence

(SEQ ID NO: 11) VDKLAAALE.

-   99. The fusion protein of any of paragraphs 82-98, wherein the    lipidated biotin-binding protein is a biotin-binding protein of any    of paragraphs 68-80.-   100. The fusion protein of any of paragraphs 82-99, wherein the    fusion protein comprises the amino acid sequence selected from the    group consisting of

(i) SEQ ID NO: 53 MKKVAAFVALSLLMAGCVSPDFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNAGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTNVDKLAAALEHHHHHH; (ii) SEQ ID NO: 54MKKVAAFVALSLLMAGCVSPDFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSANPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTNVDKLAAALEHHHHHH; and (iii) SEQ ID NO: 55 (SEQ ID NO: 55)MKKVAAFVALSLLMAGCVSPDFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYAAASWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTNVDKLAAALEHHHHHH.

-   101. A composition comprising a lipidated biotin-binding protein of    any of paragraphs 82-100.-   102. A method inducing an immune response in a subject, comprising    administering to the subject a composition of paragraph 81 or 101.-   103. A method of vaccinating a mammal against at least one    antigen-bearing pathogen, the method comprising administering a    composition of paragraph 81 or 101.-   104. The method of any of paragraphs 102 or 103, wherein the subject    is a human.-   105. The method of any of paragraphs 102 or 103, wherein the subject    is an agricultural or non-domestic animal.-   106. The method of any of paragraphs 102 or 103, wherein the subject    is a domestic animal.-   107. The method of any of paragraphs 102 or 103, wherein    administration is via subcutaneous, intranasal, intradermal or intra    muscular injection.-   108. The method of paragraph 102, wherein the immune response is an    antibody/B-cell response.-   109. The method of paragraph 102, wherein the immune response is a    CD4+ T-cell response, including Th1, Th2, or Th17 response.-   110. The method of paragraph 102, wherein the immune response is a    CD8+ T-cell response.-   111. The composition of any of paragraphs 81 or 101 for use in a    diagnostic for exposure to a pathogen or immune threat.    Some Selected Definitions

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.The term “or” is inclusive unless modified, for example, by “either.”Other than in the operating examples, or where otherwise cindicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

The term “immunogenic composition” used herein is defined as acomposition capable of eliciting an immune response, such as an antibodyor cellular immune response, when administered to a subject. Theimmunogenic compositions of the present invention may or may not beimmunoprotective or therapeutic. When the immunogenic compositions ofthe present invention prevent, ameliorate, palliate or eliminate diseasefrom the subject, then the immunogenic composition may optionally bereferred to as a vaccine. As used herein, however, the term immunogeniccomposition is not intended to be limited to vaccines.

As used herein, the term “antigen” refers to any substance that promptsan immune response directed against the substance. In some embodiments,an antigen is a peptide or a polypeptide, and in other embodiments, itcan be any chemical or moiety, e.g., a carbohydrate, that elicits animmune response directed against the substance.

The term “associates” as used herein refers to the linkage of two ormore molecules by non-covalent or covalent bonds. In some embodiments,where linking of two or more molecules occurs by a covalent bond, thetwo or more molecules can be fused together, or cross-linked together.In some embodiments, where linking of two or more molecules occurs by anon-covalent bond, the two or more molecules can form a complex.

The term “complex” as used herein refers to a collection of two or moremolecules, connected spatially by means other than a covalentinteraction; for example they can be connected by electrostaticinteractions, hydrogen bound or by hydrophobic interactions (i.e., vander Waals forces).

As used herein, the term “fused” means that at least one protein orpeptide is physically associated with a second protein or peptide. Insome embodiments, fusion is typically a covalent linkage, however, othertypes of linkages are encompassed in the term “fused” include, forexample, linkage via an electrostatic interaction, or a hydrophobicinteraction and the like. Covalent linkage can encompass linkage as afusion protein or chemically coupled linkage, for example via adisulfide bound formed between two cysteine residues.

As used herein, the term “fusion polypeptide” or “fusion protein” meansa protein created by joining two or more polypeptide sequences together.The fusion polypeptides encompassed in this invention includetranslation products of a chimeric gene construct that joins the DNAsequences encoding one or more antigens, or fragments or mutantsthereof, with the DNA sequence encoding a second polypeptide to form asingle open-reading frame. In other words, a “fusion polypeptide” or“fusion protein” is a recombinant protein of two or more proteins whichare joined by a peptide bond. In some embodiments, the second protein towhich the antigens are fused to is a complementary affinity moleculewhich is capable of interacting with a first affinity molecule of thecomplementary affinity pair.

The terms “polypeptide” and “protein” can be used interchangeably torefer to a polymer of amino acid residues linked by peptide bonds, andfor the purposes of the claimed invention, have a typical minimum lengthof at least 25 amino acids. The term “polypeptide” and “protein” canencompass a multimeric protein, e.g., a protein containing more than onedomain or subunit. The term “peptide” as used herein refers to asequence of peptide bond-linked amino acids containing less than 25amino acids, e.g., between about 4 amino acids and 25 amino acids inlength. Proteins and peptides can be composed of linearly arranged aminoacids linked by peptide bonds, whether produced biologically,recombinantly, or synthetically and whether composed of naturallyoccurring or non-naturally occurring amino acids, are included withinthis definition. Both full-length proteins and fragments thereof greaterthan 25 amino acids are encompassed by the definition of protein. Theterms also include polypeptides that have co-translational (e.g., signalpeptide cleavage) and post-translational modifications of thepolypeptide, such as, for example, disulfide-bond formation,glycosylation, acetylation, phosphorylation, lipidation, proteolyticcleavage (e.g., cleavage by metalloproteases), and the like.Furthermore, as used herein, a “polypeptide” refers to a protein thatincludes modifications, such as deletions, additions, and substitutions(generally conservative in nature as would be known to a person in theart) to the native sequence, as long as the protein maintains thedesired activity. These modifications can be deliberate, as throughsite-directed mutagenesis, or can be accidental, such as throughmutations of hosts that produce the proteins, or errors due to PCRamplification or other recombinant DNA methods.

By “signal sequence” is meant a nucleic acid sequence which, whenoperably linked to a nucleic acid molecule, facilitates secretion of theproduct (e.g., protein or peptide) encoded by the nucleic acid molecule.In some embodiments, the signal sequence is preferably located 5′ to thenucleic acid molecule.

As used herein, the term “N-glycosylated” or “N-glycosylation” refers tothe covalent attachment of a sugar moiety to asparagine residues in apolypeptide. Sugar moieties can include but are not limited to glucose,mannose, and N-acetylglucosamine. Modifications of the glycans are alsoincluded, e.g., siaylation.

An “antigen presenting cell” or “APC” is a cell that expresses the MajorHistocompatibility complex (MHC) molecules and can display foreignantigen complexed with MHC on its surface. Examples of antigenpresenting cells are dendritic cells, macrophages, B-cells, fibroblasts(skin), thymic epithelial cells, thyroid epithelial cells, glial cells(brain), pancreatic beta cells, and vascular endothelial cells.

The term “functional portion” or “functional fragment” as used in thecontext of a “functional portion of an antigen” refers to a portion ofthe antigen or antigen polypeptide that mediates the same effect as thefull antigen moiety, e.g., elicits an immune response in a subject, ormediates an association with other molecule, e.g., comprises at least onepitope.

A “portion” of a target antigen as that term is used herein will be atleast 3 amino acids in length, and can be, for example, at least 6, atleast 8, at least 10, at least 14, at least 16, at least 17, at least18, at least 19, at least 20 or at least 25 amino acids or greater,inclusive.

The terms “Cytotoxic T Lymphocyte” or “CTL” refers to lymphocytes whichinduce apoptosis in targeted cells. CTLs form antigen-specificconjugates with target cells via interaction of TCRs with processedantigen (Ag) on target cell surfaces, resulting in apoptosis of thetargeted cell. Apoptotic bodies are eliminated by macrophages. The term“CTL response” is used to refer to the primary immune response mediatedby CTL cells.

The term “cell mediated immunity” or “CMI” as used herein refers to animmune response that does not involve antibodies or complement butrather involves the activation of, for example, macrophages, naturalkiller cells (NK), antigen-specific cytotoxic T-lymphocytes (T-cells),and the release of various cytokines in response to a target antigen.Stated another way, CMI refers to immune cells (such as T cells andother lymphocytes) which bind to the surface of other cells that displaya target antigen (such as antigen presenting cells (APS)) and trigger aresponse. The response may involve either other lymphocytes and/or anyof the other white blood cells (leukocytes) and the release ofcytokines. Cellular immunity protects the body by: (i) activatingantigen-specific cytotoxic T-lymphocytes (CTLs) that are able to destroybody cells displaying epitopes of foreign antigen on their surface, suchas virus-infected cells and cells with intracellular bacteria; (2)activating macrophages and NK cells, enabling them to destroyintracellular pathogens; and (3) stimulating cells to secrete a varietyof cytokines that influence the function of other cells involved inadaptive immune responses and innate immune responses.

The term “immune cell” as used herein refers to any cell which canrelease a cytokine in response to a direct or indirect antigenicstimulation. Included in the term “immune cells” herein are lympocytes,including natural killer (NK) cells, T-cells (CD4+ and/or CD8+ cells),B-cells, macrophages and monocytes, Th cells; Th1 cells; Th2 cells;leukocytes; dendritic cells; macrophages; mast cells and monocytes andany other cell which is capable of producing a cytokine molecule inresponse to direct or indirect antigen stimulation. Typically, an immunecell is a lymphocyte, for example a T-cell lymphocyte.

The term “cytokine” as used herein refers to a molecule released from animmune cell in response to stimulation with an antigen. Examples of suchcytokines include, but are not limited to: GM-CSF; IL-1α; IL-1β; IL-2;IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12; IL-17A, IL-17F orother members of the IL-17 family, IL-22, IL-23, IFN-α; IFN-β; IFN-γ;MIP-1α; MIP-1β; TGF-β; TNFα, or TNFβ. The term “cytokine” does notinclude antibodies

The term “subject” as used herein refers to any animal in which it isuseful to elicit an immune response. The subject can be a wild,domestic, commercial or companion animal such as a bird or mammal. Thesubject can be a human. Although in one embodiment of the invention itis contemplated that the immunogenic compositions as disclosed hereincan also be suitable for the therapeutic or preventative treatment inhumans, it is also applicable to warm-blooded vertebrates, e.g.,mammals, such as non-human primates, (particularly higher primates),sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat,rabbits, cows, and non-mammals such as chickens, ducks, or turkeys. Inanother embodiment, the subject is a wild animal, for example a birdsuch as for the diagnosis of avian flu. In some embodiments, the subjectis an experimental animal or animal substitute as a disease model. Thesubject may be a subject in need of veterinary treatment, whereeliciting an immune response to an antigen is useful to prevent adisease and/or to control the spread of a disease, for example SIV,STL1, SFV, or in the case of live-stock, hoof and mouth disease, or inthe case of birds Marek's disease or avian influenza, and other suchdiseases.

As used herein, the term “pathogen” refers to an organism or moleculethat causes a disease or disorder in a subject. For example, pathogensinclude but are not limited to viruses, fungi, bacteria, parasites, andother infectious organisms or molecules therefrom, as well astaxonomically related macroscopic organisms within the categories algae,fungi, yeast, protozoa, or the like.

A “cancer cell” refers to a cancerous, pre-cancerous, or transformedcell, either in vivo, ex vivo, or in tissue culture, that hasspontaneous or induced phenotypic changes that do not necessarilyinvolve the uptake of new genetic material. Although transformation canarise from infection with a transforming virus and incorporation of newgenomic nucleic acid, or uptake of exogenous nucleic acid, it can alsoarise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation/cancer is associated with,e.g., morphological changes, immortalization of cells, aberrant growthcontrol, foci formation, anchorage independence, malignancy, loss ofcontact inhibition and density limitation of growth, growth factor orserum independence, tumor specific markers, invasiveness or metastasis,and tumor growth in suitable animal hosts such as nude mice. See, e.g.,Freshney, CULTURE ANIMAL CELLS: MANUAL BASIC TECH. (3rd ed., 1994).

The term “wild type” refers to the naturally-occurring, normalpolynucleotide sequence encoding a protein, or a portion thereof, orprotein sequence, or portion thereof, respectively, as it normallyexists in vivo.

The term “mutant” refers to an organism or cell with any change in itsgenetic material, in particular a change (i.e., deletion, substitution,addition, or alteration) relative to a wild-type polynucleotide sequenceor any change relative to a wild-type protein sequence. The term“variant” may be used interchangeably with “mutant”. Although it isoften assumed that a change in the genetic material results in a changeof the function of the protein, the terms “mutant” and “variant” referto a change in the sequence of a wild-type protein regardless of whetherthat change alters the function of the protein (e.g., increases,decreases, imparts a new function), or whether that change has no effecton the function of the protein (e.g., the mutation or variation issilent).

The term “pharmaceutically acceptable” refers to compounds andcompositions which may be administered to mammals without unduetoxicity. The term “pharmaceutically acceptable carriers” excludestissue culture medium. Exemplary pharmaceutically acceptable saltsinclude but are not limited to mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like, andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Pharmaceutically acceptable carriers arewell-known in the art.

It will be appreciated that proteins or polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, can be modified in a given polypeptide, eitherby natural processes such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Known modifications which can be present inpolypeptides of the present invention include, but are not limited to,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa polynucleotide or polynucleotide derivative, covalent attachment of alipid or lipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formulation, gamma-carboxylation, glycation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination.

As used herein, the terms “homologous” or “homologues” are usedinterchangeably, and when used to describe a polynucleotide orpolypeptide, indicate that two polynucleotides or polypeptides, ordesignated sequences thereof, when optimally aligned and compared, forexample using BLAST, version 2. 2. 14 with default parameters for analignment are identical, with appropriate nucleotide insertions ordeletions or amino-acid insertions or deletions, in at least 70% of thenucleotides, usually from about 75% to 99%, such as at least about 98 to99% of the nucleotides. For a polypeptide, there should be at least 50%of amino acid identity in the polypeptide. The term “homolog” or“homologous” as used herein also refers to homology with respect tostructure. Determination of homologs of genes or polypeptides can beeasily ascertained by the skilled artisan. When in the context with adefined percentage, the defined percentage homology means at least thatpercentage of amino acid similarity. For example, 85% homology refers toat least 85% of amino acid similarity.

As used herein, the term “heterologous” reference to nucleic acidsequences, proteins or polypeptides mean that these molecules are notnaturally occurring in that cell. For example, the nucleic acid sequencecoding for a fusion antigen polypeptide described herein that isinserted into a cell, e.g. in the context of a protein expressionvector, is a heterologous nucleic acid sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters. Where necessary or desired, optimalalignment of sequences for comparison can be conducted by any variety ofapproaches, as these are well-known in the art.

The term “variant” as used herein may refer to a polypeptide or nucleicacid that differs from the naturally occurring polypeptide or nucleicacid by one or more amino acid or nucleic acid deletions, additions,substitutions or side-chain modifications, yet retains one or morespecific functions or biological activities of the naturally occurringmolecule. Amino acid substitutions include alterations in which an aminoacid is replaced with a different naturally-occurring or anon-conventional amino acid residue. Such substitutions may beclassified as “conservative,” in which case an amino acid residuecontained in a polypeptide is replaced with another naturally occurringamino acid of similar character either in relation to polarity, sidechain functionality or size. Substitutions encompassed by variants asdescribed herein may also be “non conservative,” in which an amino acidresidue which is present in a peptide is substituted with an amino acidhaving different properties (e.g., substituting a charged or hydrophobicamino acid with alanine), or alternatively, in which anaturally-occurring amino acid is substituted with a non-conventionalamino acid. Also encompassed within the term “variant,” when used withreference to a polynucleotide or polypeptide, are variations in primary,secondary, or tertiary structure, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild-type polynucleotide or polypeptide).

The term “substantially similar,” when used in reference to a variant ofan antigen or a functional derivative of an antigen as compared to theoriginal antigen means that a particular subject sequence varies fromthe sequence of the antigen polypeptide by one or more substitutions,deletions, or additions, but retains at least 50%, or higher, e.g., atleast 60%, 70%, 80%, 90% or more, inclusive, of the function of theantigen to elicit an immune response in a subject. In determiningpolynucleotide sequences, all subject polynucleotide sequences capableof encoding substantially similar amino acid sequences are considered tobe substantially similar to a reference polynucleotide sequence,regardless of differences in codon sequence. A nucleotide sequence is“substantially similar” to a given antigen nucleic acid sequence if: (a)the nucleotide sequence hybridizes to the coding regions of the nativeantigen sequence, or (b) the nucleotide sequence is capable ofhybridization to nucleotide sequence of the native antigen undermoderately stringent conditions and has biological activity similar tothe native antigen protein; or (c) the nucleotide sequences aredegenerate as a result of the genetic code relative to the nucleotidesequences defined in (a) or (b). Substantially similar proteins willtypically be greater than about 80% similar to the correspondingsequence of the native protein.

Variants can include conservative or non-conservative amino acidchanges, as described below. Polynucleotide changes can result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence. Variants can also includeinsertions, deletions or substitutions of amino acids, includinginsertions and substitutions of amino acids and other molecules) that donot normally occur in the peptide sequence that is the basis of thevariant, for example but not limited to insertion of ornithine which donot normally occur in human proteins. “Conservative amino acidsubstitutions” result from replacing one amino acid with another havingsimilar structural and/or chemical properties. Conservative substitutiontables providing functionally similar amino acids are well known in theart. For example, the following six groups each contain amino acids thatare conservative substitutions for one another: (1) Alanine (A), Serine(S), Threonine (T); (2) Aspartic acid (D), Glutamic acid (E); (3)Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and (6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See, e.g., Creighton,PROTEINS (W. H. Freeman & Co., 1984).

The choice of conservative amino acids may be selected based on thelocation of the amino acid to be substituted in the peptide, for exampleif the amino acid is on the exterior of the peptide and exposed tosolvents, or on the interior and not exposed to solvents. Selection ofsuch conservative amino acid substitutions is within the skill of one ofordinary skill in the art. Accordingly, one can select conservativeamino acid substitutions suitable for amino acids on the exterior of aprotein or peptide (i. e. amino acids exposed to a solvent). Thesesubstitutions include, but are not limited to the following:substitution of Y with F, T with S or K, P with A, E with D or Q, N withD or G, R with K, G with N or A, T with S or K, D with N or E, I with Lor V, F with Y, S with T or A, R with K, G with N or A, K with R, A withS, K or P.

Alternatively, one can also select conservative amino acid substitutionssuitable for amino acids on the interior of a protein or peptide (i.e.,the amino acids are not exposed to a solvent). For example, one can usethe following conservative substitutions: where Y is substituted with F,T with A or S, I with L or V, W with Y, M with L, N with D, G with A, Twith A or S, D with N, I with L or V, F with Y or L, S with A or T and Awith S, G, T or V. In some embodiments, LF polypeptides includingnon-conservative amino acid substitutions are also encompassed withinthe term “variants.” As used herein, the term “non-conservative”substitution refers to substituting an amino acid residue for adifferent amino acid residue that has different chemical properties.Non-limiting examples of non-conservative substitutions include asparticacid (D) being replaced with glycine (G); asparagine (N) being replacedwith lysine (K); and alanine (A) being replaced with arginine (R).

The term “derivative” as used herein refers to peptides which have beenchemically modified, for example by ubiquitination, labeling, pegylation(derivatization with polyethylene glycol) or addition of othermolecules. A molecule is also a “derivative” of another molecule when itcontains additional chemical moieties not normally a part of themolecule. Such moieties can improve the molecule's solubility,absorption, biological half-life, etc. The moieties can alternativelydecrease the toxicity of the molecule, or eliminate or attenuate anundesirable side effect of the molecule, etc. Moieties capable ofmediating such effects are disclosed in REMINGTON'S PHARMACEUTICALSCIENCES (21st ed., Tory, ed., Lippincott Williams & Wilkins, Baltimore,Md., 2006).

The term “functional” when used in conjunction with “derivative” or“variant” refers to a protein molecule which possesses a biologicalactivity that is substantially similar to a biological activity of theentity or molecule of which it is a derivative or variant.“Substantially similar” in this context means that the biologicalactivity, e.g., antigenicity of a polypeptide, is at least 50% as activeas a reference, e.g., a corresponding wild-type polypeptide, e.g., atleast 60% as active, 70% as active, 80% as active, 90% as active, 95% asactive, 100% as active or even higher (i.e., the variant or derivativehas greater activity than the wild-type), e.g., 110% as active, 120% asactive, or more, inclusive.

The term “recombinant” when used to describe a nucleic acid molecule,means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/orsynthetic origin, which, by virtue of its origin or manipulation, is notassociated with all or a portion of the polynucleotide sequences withwhich it is associated in nature. The term recombinant as used withrespect to a peptide, polypeptide, protein, or recombinant fusionprotein, means a polypeptide produced by expression from a recombinantpolynucleotide. The term recombinant as used with respect to a host cellmeans a host cell into which a recombinant polynucleotide has beenintroduced. Recombinant is also used herein to refer to, with referenceto material (e.g., a cell, a nucleic acid, a protein, or a vector) thatthe material has been modified by the introduction of a heterologousmaterial (e.g., a cell, a nucleic acid, a protein, or a vector).

The term “vectors” refers to a nucleic acid molecule capable oftransporting or mediating expression of a heterologous nucleic acid towhich it has been linked to a host cell; a plasmid is a species of thegenus encompassed by the term “vector.” The term “vector” typicallyrefers to a nucleic acid sequence containing an origin of replicationand other entities necessary for replication and/or maintenance in ahost cell. Vectors capable of directing the expression of genes and/ornucleic acid sequence to which they are operatively linked are referredto herein as “expression vectors”. In general, expression vectors ofutility are often in the form of “plasmids” which refer to circulardouble stranded DNA molecules which, in their vector form are not boundto the chromosome, and typically comprise entities for stable ortransient expression or the encoded DNA. Other expression vectors thatcan be used in the methods as disclosed herein include, but are notlimited to plasmids, episomes, bacterial artificial chromosomes, yeastartificial chromosomes, bacteriophages or viral vectors, and suchvectors can integrate into the host's genome or replicate autonomouslyin the particular cell. A vector can be a DNA or RNA vector. Other formsof expression vectors known by those skilled in the art which serve theequivalent functions can also be used, for example self replicatingextrachromosomal vectors or vectors which integrates into a host genome.Preferred vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked.

The term “reduced” or “reduce” or “decrease” as used herein generallymeans a decrease by a statistically significant amount relative to areference. For avoidance of doubt, “reduced” means statisticallysignificant decrease of at least 10% as compared to a reference level,for example a decrease by at least 20%, at least 30%, at least 40%, atleast t 50%, or least 60%, or least 70%, or least 80%, at least 90% ormore, up to and including a 100% decrease (i.e., absent level ascompared to a reference sample), or any decrease between 10-100% ascompared to a reference level, as that term is defined herein.

The term “low” as used herein generally means lower by a staticallysignificant amount; for the avoidance of doubt, “low” means astatistically significant value at least 10% lower than a referencelevel, for example a value at least 20% lower than a reference level, atleast 30% lower than a reference level, at least 40% lower than areference level, at least 50% lower than a reference level, at least 60%lower than a reference level, at least 70% lower than a reference level,at least 80% lower than a reference level, at least 90% lower than areference level, up to and including 100% lower than a reference level(i.e., absent level as compared to a reference sample).

The terms “increased” or “increase” as used herein generally mean anincrease by a statically significant amount; such as a statisticallysignificant increase of at least 10% as compared to a reference level,including an increase of at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100% or more, inclusive, including, for example at least 2-fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 10-foldincrease or greater as compared to a reference level, as that term isdefined herein.

The term “high” as used herein generally means a higher by a staticallysignificant amount relative to a reference; such as a statisticallysignificant value at least 10% higher than a reference level, forexample at least 20% higher, at least 30% higher, at least 40% higher,at least 50% higher, at least 60% higher, at least 70% higher, at least80% higher, at least 90% higher, at least 100% higher, inclusive, suchas at least 2-fold higher, at least 3-fold higher, at least 4-foldhigher, at least 5-fold higher, at least 10-fold higher or more, ascompared to a reference level.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described herein.

As used herein the term “biotin” refers to the compound biotin itselfand analogues, derivatives and variants thereof. Thus, the term “biotin”includes biotin (cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid) and any derivatives and analogsthereof, including biotin-like compounds. Such compounds include, forexample, biotin-e-N-lysine, biocytin hydrazide, amino or sulfhydrylderivatives of 2-iminobiotin and biotinyl-E-aminocaproicacid-N-hydroxysuccinimide ester, sulfosuccinimideiminobiotin,biotinbromoacetylhydrazide, p-diazobenzoyl biocytin,3-(N-maleimidopropionyObiocytin, desthiobiotin, and the like. The term“biotin” also comprises biotin variants that can specifically bind toone or more of a Rhizavidin, avidin, streptavidin, tamavidin moiety, orother avidin-like peptides.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity of understandingit will be readily apparent to one of ordinary skill in the art in lightof the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims. The following is meant to be illustrativeof the present invention; however, the practice of the invention is notlimited or restricted in any way by the examples.

EXAMPLES Example 1: Expressing High Yield and Soluble RecombinantBiotin-Binding Protein and Fusion Proteins Thereof in E. coli

The recombinant Rhizavidin (rRhavi) used in these studies is anN-terminal modified version that contains only the residues 45 to 179 ofthe wild type protein. To optimize the expression level of rRhavi in E.coli, the gene sequence that encodes Rhizavidin polypeptides (45-179)was re-designed by using E. coli-preferred expression codons, thensynthesized and cloned into the PET21b vector. To facilitate the correctfolding and obtain a high yield of soluble recombinant protein, a DNAsequence encoding an E. coli periplasmic localization signal sequence(19 amino acids, MKKIWLALAGLVLAFSASA, SEQ ID NO: 2) was introduced atthe 5′ end of the synthetic gene of rRhavi. This signal sequence ispredicted to be deleted automatically from the recombinant protein afterits targeting to the periplasm of E. coli during the process ofexpression.

A DNA sequence encoding a flexible linker region and His-tag(GGGGSSSVDKLAAALEHHHHHH, SEQ ID NO: 14) was directly inserted into the3′ end of the synthetic rRhavi gene. This helps for the purification ofrecombinant biotin-binding protein. Furthermore, an antigen can beinserted in the linker having a flexible linker on both sides, e.g., theantigen can be inserted between amino acids S and V of the linker. Assuch the antigen is separated from the biotin-binding protein by thepeptide linker (GGGGSSS, SEQ ID NO: 22) and from the His-tag by thepeptide linker (VDKLAAALE, SEQ ID NO: 11) this can stabilize the fusionprotein.

To construct Rhizavidin-antigen fusion proteins, a DNA sequence encodinga flexible linker region consisting of seven amino acids (GGGGSSS, SEQID NO: 22) can be directly inserted into the 3′ end of the syntheticrRhavi gene, to help stabilize the fusion protein. The genes encodingcandidate antigens (full length or desired fragment) were amplified fromthe genomic DNA of interested pathogens by routine PCR procedures andinserted into the rRhavi expression vector just beyond the linkerregion.

For protein expression, the plasmids containing target constructs weretransformed into E. coli strain BL21 (DE3) using standard heat-shockprocedure. A single colony was picked freshly from the plate (or aglycerol stock was used later) and inoculated into 30 ml Luria-Bertani(LB) medium containing Ampicillin (Amp+) for an overnight culture at 37°C. On day 2, a 5 ml starting culture was inoculated into 1 liter of LBmedium/Amp+ and grown at 37° C. until OD₆₀₀=1 was reached. After coolingthe medium to 16° C., 0.2 mM final concentration of IPTG was added intothe cultures for an overnight induction.

Proteins were purified from the periplasmic fraction using a modifiedosmotic shock protocol. Briefly, the bacterial cells from the 6 literculture were collected and re-suspended in 120 ml buffer containing 30mM Tris (pH 8.0), 20% sucrose and 1 mM EDTA. After stirring at roomtemperature for 20 min, the cells were re-pelleted by centrifugation at10,000 rpm for 10 min. The supernatant was collected as fraction 1, andthe cells were re-suspended in 80 ml ice cold solution containing 5 mMMgCl2, proteinase inhibitor and DNase. After stirring at 4° for 20 min,the mixture was subjected to centrifugation at 13,000 rpm for 20 min andthe supernatant was collected as fraction 2. After adding a finalconcentration of 150 mM NaCl, 10 mM MgCl₂ and 10 mM Imidazole, thesupernatant combining fraction 1 and fraction 2 was applied onto aNi-NTA column. The proteins eluted from the Ni-NTA column were furtherpurified by gel-filtration using superdex 200 column running on AKTApurifier. The peak fractions containing target protein were pooled andconcentrated. The protein concentration was measured by using BCAprotein assay kit from Bio-Rad. Purified proteins were aliquoted,flash-frozen in liquid nitrogen and kept at −80° C. for future use.

The construct of biotin-binding protein is shown schematically in FIG. 1, and SDS-PAGE of the purified biotin-binding protein is shown in FIG. 2.

The construct of fusion proteins comprising biotin-binding protein isshown schematically in FIG. 3 , and the exemplary SDS-PAGE of thepurified fusion proteins are shown in FIG. 4 .

Example 2: Lipidated Derivative of Biotin-Binding Proteins

A lipidated derivate of recombinant biotin-binding protein was producedusing a method similar to the one described in Example 1. The lipidatedderivate used in this study is an N-terminal modified version of wildtype Rhizavidin that contains only the residues 45 to 179 of the wildtype protein. To optimize the expression level of rRhavi in E. coli, thegene sequence that encodes Rhizavidin polypeptides (45-179) wasre-designed by using E. coli preferred expression codons, thensynthesized and cloned into the PET2 lb vector. To facilitate thelipidation, correct folding and obtain a high yield of solublerecombinant protein, a DNA sequence encoding lipidation sequence (19amino acids, MKKVAAFVALSLLMAGC, SEQ ID NO: 3) was introduced at the 5′end of the synthetic gene of rRhavi. The lipidation will be added on theCys residue of lipidation sequence by bacreria, e.g., E. coli, duringthe process of expression.

For protein expression, the plasmid containing target constructs wastransformed into E. coli strain BL21 (DE3) using standard heat-shockprocedure. A single colony was picked freshly from the plate (or aglycerol stock was used later) and inoculated into 30 ml Luria-Bertani(LB) medium containing Ampicillin (Amp+) for an overnight culture at 37°C. On day 2, a 5 ml starting culture was inoculated into 1 liter of LBmedium/Amp+ and grown at 37° C. until OD₆₀₀=1 was reached. After coolingthe medium to 16° C., 0.2 mM final concentration of IPTG was added intothe cultures for an overnight induction.

Lipidated rhizavidin was purified from E. coli membrane franction. Ecoli cells were collected and resuspended in lysis buffer (20 mM Tris,500 mM NaCl, pH 8.0) containing protease inhibitors, Dnase, 10 mM Mg²⁺and lysozyme. Cells were disrupted by one freeze-thaw cycle and thesupernatant was removed after centrifugation at 13,000 rpm for 45 min.Cell pellets were then resuspended in lysis buffer containing 0.5% SDOC,and homogenized by beads beater. The lysates were then applied forcentrifugation at 13,000 rpm for 45 min, and the supernatant wascollected for affinity purification. Lipidated rhavi was eluted withlysis buffer containing 0.5% SDOC and 300 mM Im.

The proteins eluted from the Ni-NTA column were further purified bygel-filtration using superdex 200 column running on AKTA purifier. Thepeak fractions containing target protein were pooled and concentrated.The protein concentration was measured by using BCA protein assay kitfrom Bio-Rad. Purified proteins were aliquoted, flash-frozen in liquidnitrogen and kept at −80° C. for future use.

The lipidated biotin-binding protein produced is shown schematically inFIG. 5 , and SDS-PAGE of the purified lipidated biotin-binding proteinis shown in FIG. 6 .

Example 3: TLR2 Activity of Lipidated Biotin-Binding Protein

TLR2 activity of lipidated biotin-binding protein was tested in HEK TLR2cells. HEK TLR2 cells were plated in 24 well plate at 5×10⁵ cells/perwell in 500 μl volume. Lipidated biotin-binding protein was added atdifferent concentrations for stimulation at 37° C. overnight. Thesupernatants were collected the second day for IL-8 measurement byELISA. As a control, HEK 293 cells were used for stimulation at the samecondition.

TLR2 activity of lipidated biotin-binding-protein was determined.Results showed that lipidated biotin-binding protein induced productionof IL-8 from HEK TLR2 but not from HEK 293 cells (FIG. 7 ).

Example 4: Non-Hemolytic Mutants of Hla and Fusion Proteins

The DNA sequence encoding wild type Hla mature polypeptide (amino acid27 to 319) was cloned from Staphylococcus aurues genome. Allnon-hemolytic mutants of Hla were generated by site-directed mutagenesisusing quickchange. To make Hla-biotin binding fusion proteins, the DNAsequence encoding wild type Hla or mutant Hla was inserted beyond thelinker region followed biotin-binding protein gene. All constructs werecloned into PET21b and transformed into E. coli for expression asdescribed above.

Non-hemolytic mutants of Hla were produced. The exemplary non-hemolyticvariants of Hla are shown schematically in FIG. 8 . SDS-PAGE of thepurified wild-type or non-hemolytic variants of Hla and fusion proteinis shown in FIGS. 9 and 10 .

Example 5: Hemolytic Activity of Wild Type Hla, Mutant Hla and FusionProteins

The hemolytic activity of wild type Hla, mutant Hla and their fusionproteins with biotin-binding protein was analyzed using rabbit bloodcells. Red blood cells from 250 μl of rabbit blood were pelleted, washedwith PBS twice and then resuspended in 10 ml of PBS. Wild type Hla,mutant Hla and fusion proteins were diluted with PBS at indicatedconcentrations and then added into 96 well plate at 100 μl per well.Blood cells were added into 96 well plate containing Hla or fusionproteins at 25 μl per well and then incubated at 37° C. for 30 min.Supernatants were collected after centrifugation at 2000 rpm for 5 minand analyzed by ELISA reader at OD450.

hemolytic activity of wild type Hla, mutant Hla and their fusionproteins was assayed. Results demonstrate that mutant Hla have muchlower hemolytic activity relative to wild-type Hla (FIG. 11 ). Further,the Hla fusion proteins comprising a biotin-binding protein had evenlower hemolytic activity relative to the non-fusion mutant Hla protein(FIG. 12 ).

Example 6: Stimulatory Activity of Mutant Hla Fusion Protein

C57 WT macrophage cells were stimulated with non-hemolytic Hla mutantfusion protein. Cells were seeded in 24 well plate at 5×10⁵ cells perwell. Mutant Hla fusion protein was diluted with growth medium and addedinto wells at indicated concentration for stimulation at 37° C.overnight. Supernatants were collected the second day aftercentrifugation at 2000 rpm for 5 min and then analyzed for cytokinesecretion by ELISA.

Stimularoty activity of mutant Hla fusion protein was analyzed. Resultsshowed that mutant Hla fusion protein (rhavi-Hla209) induced productionof multiple pro-inflammatory cytokines, including TNF-α, IL-6, Il-23,IL-1β, and IL-17 (FIG. 13 ).

Example 7. Lipidated Biotin-Binding Proteins and Mutant Hla FusionProteins Facilitate the Immune Response to Other Antigens

MAPS based vaccine constructs were made from biotinylated serotype-1pneumococcal capsular polysaccharide, rhizavidin fused TB antigens andeither one from non-lipidated rhizavidin, lipidated rhizavidin orrhavi-Hla209. Mice were immunized with different MAPS constructs and theT cell responses against TB antigens in different immunization groupswere analyzed and compared after 3 immunizations. Briefly, the wholeblood from different mice groups were stimulated with purified TBproteins in vitro at 37° C. for 6 days and the cytokine concentration inthe supernatant was detected by ELISA.

The results showed that the mice groups received MAPS complex containinglipidated rhizavidin or containing rhavi-Hla209 generated better Th17(IL-17A) and Th1 cell (IFN-γ) responses to the TB antigens (FIG. 14 ).This indicated that lipidated rhizavidin and rhavi-Hla209 can act as aco-stimulatior/adjuvant in MAPS vaccine formulation.

It is understood that the foregoing detailed description and examplesare illustrative only and are not to be taken as limitations upon thescope of the invention. Various changes and modifications to thedisclosed embodiments, which will be apparent to those of skill in theart, may be made without departing from the spirit and scope of thepresent invention. Further, all patents and other publicationsidentified are expressly incorporated herein by reference for thepurpose of describing and disclosing, for example, the methodologiesdescribed in such publications that might be used in connection with thepresent invention. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

Further, to the extent not already indicated, it will be understood bythose of ordinary skill in the art that any one of the variousembodiments herein described and illustrated can be further modified toincorporate features shown in any of the other embodiments disclosedherein.

We claim:
 1. A polypeptide comprising an alpha-hemolysin (Hla) polypeptide, comprising a mutation at amino acid residue 205, 213 or 209-211 of wild-type S. aureus alpha-hemolysin polypeptide, wherein the alpha-hemolysin polypeptide has lower hemolytic activity than an equivalent titer of wild-type Hla polypeptide.
 2. The polypeptide_of claim 1, wherein the hemolytic activity of the alpha-hemolysin polypeptide is at least 25% lower than an equivalent titer of wild-type Hla polypeptide.
 3. The polypeptide of claim 1, wherein the alpha-hemolysin polypeptide comprises one of the following mutations in the wild-type S. aureus alpha-hemolysin polypeptide: (i) residue 205 W to A; (ii) residue 213 W to A; or (iii) residues 209-211 DRD to AAA.
 4. The polypeptide of claim 1, wherein the alpha-hemolysin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO:
 25. 5. A composition comprising the polypeptide of claim
 1. 6. A method inducing an immune response in a subject, comprising administering to the subject a composition of claim
 5. 7. The method of claim 6, wherein the subject is a human.
 8. The method of claim 6, wherein the immune response is an antibody/B-cell response.
 9. The method of claim 6, wherein the immune response is a CD4+ T-cell response, including Th1, Th2, or Th17 response, and/or a CD8+ T-cell response.
 10. The polypeptide of claim 1, wherein the wild-type Hla polypeptide comprises the amino acid sequence as set forth below: (SEQ ID NO: 59) ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKK LLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYY PRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDF KTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSM KAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDD YQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN

wherein the amino acid sequence of SEQ ID NO: 59 corresponds to amino acids 27-319 of the wild-type Hla mature polypeptide.
 11. The polypeptide of claim 1, further comprising a biotin-binding domain.
 12. The polypeptide of claim 11, wherein the biotin-binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO:
 1. 13. The polypeptide of claim 11, comprising a peptide linker between the biotin-binding domain and the alpha-hemolysin polypeptide.
 14. The polypeptide of claim 13, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:
 22. 15. The polypeptide of claim 11, comprising a bacterial signal sequence at the N-terminus.
 16. The polypeptide of claim 11, comprising a purification tag at the C-terminus.
 17. A composition comprising the polypeptide of claim
 11. 18. The polypeptide of claim 11, wherein the biotin-binding domain comprises the amino acid sequence of SEQ ID NO:
 1. 